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This is to certify that the

thesis entitled

PATTERNS IN THE VOLATILE PROFILE FOR 'REDCHIEF

DELICIOUS' APPLE FRUIT DURING RIPENING
AND SENESCENCE

presented by
MARIA ALEJANDRA FERENCZI GARDINI

has been accepted towards fulﬁllment
of the requirements for

MASTER OIL—SCIENCE—degree in HORTICULTURE

JOI’ professor

 

Date5-”03

0.7639 MS U is an Afﬁrmative Action/Equal Opportunity Institution

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

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c10lRC/DateDue.p65-p.15

PATTER

PATTERNS IN THE VOLATILE PROFILE FOR ‘REDCHIEF DELICIOUS’ APPLE
FRUIT DURING RIPENING AND SENESCENCE

By

Maria Alejandra Ferenczi Gardini

A THESIS
Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Horticulture

2003

PATTERI

 

 

 

ABSTRACT

PATTERNS IN THE VOLATILE PROFILE FOR ‘REDCHIEF DELICIOUS’ APPLE
FRUIT DURING RIPENING AND SENESCENCE

BY

Maria Alejandra Ferenczi Gardini

The volatile proﬁle of apple fruit was tracked from three weeks prior to eight
weeks after the onset of the ethylene climacteric. The peak in ester emanation
roughly coincided with the maxima for respiration and ethylene production. The
esters were evaluated separately according to the acid and alcohol portion. As
ripening progressed, the chain length of the alcohol-derived portion of the
predominant ester declined. Prior to the onset of the ethylene climacteric, esters
formed with hexyl alcohol predominated. Throughout the early portion of the
climacteric, esters with butyl alcohols predominated. Esters formed with propyl
alcohols were the predominant esters during the late climacteric and early
senescence phase. In late senescence, the esters from ethyl alcohol were the
predominant esters. This pattern was not observed in the chain length of the fatty
acid portion. Acetate esters predominated prior to the climacteric and also during
the latter stages of senescence. In some cases, despite an increase in acid and
alcohol substrates availability, the associated esters declined suggesting that
there is an enzymatic factor limiting ester formation. The data suggest that the
ester precursor production is developmentally regulated throughout ripening and

senescence.

To the memory of
Dr. Albertina Guarinoni

 

I v.
knowled;
physiolog
to my idea
paﬁence '
members
the time t;

Iwe

 

Drs. Robe:
Makhedov,
VanAgtma
Maneesin,
Melissa w}
hebiencoi
this thesis,
MSU gradr
much aDDn

HOWCUIIUr;

ACKNOWLEDGMENTS

I want to thank my mentor, Dr. Randy M. Beaudry for freely giving me
knowledge, wisdom and insight. Randy not only taught me about postharvest
physiology, but also life. I sincerely thank Randy for trusting in my work, listening
to my ideas and for sharing his ideas. Randy, I really appreciate your endless
patience in answering my endless questions. I also want to thank the other
members of my committee, Drs. David Dilley and Christopher Benning for taking
the time to read this work and for their contributions.

I want to express my special thanks to all the MSU postharvest people:
Drs. Robert Hemer, Sastry Jayanty, Deirdre Holcroft, Dina Kadyrjanova, Sergei
Makhedov, Zhenyong Wang, Nazir Mir, and Ludmila Roze; Najma Mir, Ryan
VanAgtmael, Katie Schwallier, Elzette Van Rooyhen, Sukasem Sittipod, Pattra
Maneesin, Ann Clements, and particularly to John Golding, Mauricio Canoles,
Melissa Whitaker, Melissa Butkiewicz, and Brian Kevany. Thank you for your
help, encouragement and especially your friendship. I may forget some details of
this thesis, but I will never forget any of you. Many thanks also to all the other
MSU graduate students. These are my friends from all over the world. I also very
much appreciate the help and support of all the staff and faculty of the MSU
Horticultural Department.

Thanks also to the Latino American Community that made us to feel
welcome at MSU and not so far away from home. Thanks particularly to the

Marquez family, Mari Paz Gonzalez and Luis Flores. But most of all I am

elemali‘,“
adorable

ready to '

 

l c:
colleague
Tabare A:
Telias an:

Masters tr

   
  
  
  

Vet
PUPPO, ar.
postharves

Tha.

 

eternally grateful to the Uruguayans; Ernesto and Daniela Restaino and their
adorable children Joaquin and Silvina, and to Jorge Arboleya, who were always
ready to help!

I owe a signiﬁcant debt of gratitude as well to all my work mates and
colleagues from the School of Agronomy in Uruguay, including Luis Viega,
Tabare Abadie, Mercedes Arias, Natalia Olivo, Giuliana Gambetta, Adriana
Telias and the rest of the crew, for their support and encouragement to make this
Masters thesis possible.

Very special thanks also to Dr.Albertina Guarinoni, Ing. Agr. Andres
Puppo, and Dr. Marius Huysamer, who introduced me to the brave new terrain of
postharvest physiology.

Thank you to all my friends, especially to Mariana Cattaneo, Andrea
Pastore, Gretel Ruprechter and Fernanda Skowronek for our continuing fresh
friendship while being so far away from each other.

I especially want to thank my large extended family, especially my Mum
and Dad, Nilda and Roberto, my brother and sister, Arturo and Elisa, and my
Mother in law, Cristina, for their patience, continuing support, constant
encouragement and love. And a very special thanks to Harry, my Father in law, I
am sure you are very happy and proud of my work.

And ﬁnally my loving husband Robert. Robert’s endless, dedicated love
and boundless support has been of constant inspiration in my life and work.
Words cannot thank you enough. And ﬁnally Harrycito, our little one, he truly

helped Mom during the last 40 weeks of this work!. THANK YOU!!

LIST OF
LIST OF
CHAPTE?‘

lNTRODLi
Re

 

   

CHAPTEF
LITERATI

TABLE OF CONTENTS

LIST OF TABLES ............................................... vii

LIST OF FIGURES ............................................... xi

CHAPTER 1

INTRODUCTION ................................................ 1
References ............................................... 5

CHAPTER 2

LITERATURE REVIEW ........................................... 6
Apple volatiles during fruit development and ripening ............... 8
Ester biosynthesis ......................................... 12
Preharvest factors affecting aroma biosynthesis .................. 20
Postharvest factors affecting aroma biosynthesis ................. 22
Measuring the volatile components of apple fruit .................. 25
References .............................................. 28

CHAPTER 3

PATTERNS IN THE ALCOHOL PORTION OF ESTERS PRODUCED DURING
RIPENING AND SENESCENCE OF ‘REDCHIEF DELICIOUS’ APPLE FRUIT 36

Introduction .............................................. 37

Materials and Methods ...................................... 39

Results .................................................. 42

Discussion ............................................... 44

Conclusions .............................................. 48

References .............................................. 49
CHAPTER 4

PATTERNS IN THE ACID PORTION OF ESTERS PRODUCED DURING
RIPENING AND SENESCENCE OF ‘REDCHIEF DELICIOUS’ APPLE FRUIT. 76

Introduction .............................................. 77
Materials and Methods ...................................... 79
Results .................................................. 82
Discussion ............................................... 84
Conclusions .............................................. 87
References .............................................. 88
APPENDIX ................................................... 1 09

vi

 

CHAPTE

Table 1.

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Table 2. \
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Table 3, C
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APPENDI.

 

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Table 4. o
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Table 7. 0ft

LIST OF TABLES

CHAPTER 3

Table 1. Matrix of target esters based on acid and alcohol precursors for apple
fruit. Detected esters are indicated. Esters in bold type were quantiﬁed
using gas standards. ...................................... 52

Table 2. Volatile compounds (esters, alcohols, and acids) identiﬁed in ‘Redchief
Delicious’ apple fruit during ripening and senescence as a function of GC
retention time (seconds). ................................... 53

Table 3. Conversion factor to calculate the concentration of those volatile
compounds present in the standard. .......................... 55

APPENDIX

Table 1. Original data of Figure 2 (Chapter 3). Ethylene content and CO2
production. Each value is the average of 5 replications. ......... 110

Table 2. Original data of Figure 3a and 3b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each hexanoate ester during
ripening and senescence. Each value is the average of ﬁve
replications. ............................................ 1 1 1

Table 3. Original data of Figure 30 (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each hexanoate ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 113

Table 4. Original data of Figure 4a and 4b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each butanoate ester during
ripening and senescence. Each value is the average of ﬁve
replications. ........................................... 1 15

Table 5. Original data 'of Figure 4c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each butanoate ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 117

Table 6. Original data of Figure 5a and 5b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each propanoate ester during
ripening and senescence. Each value is the average of ﬁve replications.

...................................................... 1 18

Table 7. Original data of Figure 5c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each propanoate ester during ripening and

vii

Table 8,

Table 9.
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ll

senescence. Each value is the average of ﬁve replications. ....... 120

Table 8. Original data of Figure 6a and 6b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each acetate ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 122

Table 9. Original data of Figure 6c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each acetate ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 124

Table 10. Original data of Figure 7a and 7b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each 2-methylbutanoate ester
during ripening and senescence. Each value is the average of ﬁve
replications. ............................................ 126

Table 11. Original data of Figure 7c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each 2-methylbutanoate ester during

ripening and senescence. Each value is the average of ﬁve replications.
...................................................... 1 28

Table 12. Original data of Figure 8a and 8b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each pentanoate ester during

ripening and senescence. Each value is the average of ﬁve replications.
....................................................... 129

Table 13. Original data of Figure 8c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each pentanoate ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 130

Table 14. Original data of Figure 93 and 9b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each heptanoate ester during
ripening and senescence. Each value is the average of ﬁve replications.
....................................................... 1 31

Table 15. Original data of Figure 9c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each heptanoate ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 132

Table 16. Original data of Figure 10a and 10b (chapter 3). Absolute, corrected
GCIMS response (total ion counts) of each octanoate ester during

ripening and senescence. Each value is the average of ﬁve replications.
....................................................... 1 33

Table 17. Original data of Figure 10c (chapter 3). Fractions, corrected GCIMS
response (total ion counts) of each octanoate ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 134

Table 18. Original data of Figure 11 (chapter 3). Absolute, corrected GCIMS

viii

 

 

Table 21
re
se

Table 22.
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ar

 

Table 23,
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se

Table 24.

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Table 25. .

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Table 28 C
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Table 29. O

resp
and

response (total ion counts) of each alcohol during ripening and
senescence. Each value is the average of ﬁve replications. ....... 135

Table 19. Original data of Figure 12 (chapter 3). Concentration (ppm) of esters
and alcohols during ripening and senescence. Each value is the average
of ﬁve replications. ....................................... 136

Table 20. Original data of Figure 1a and 1b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each hexyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 137

Table 21. Original data of Figure 1c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each hexyl ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 139

Table 22. Original data of Figure 2a and 2b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each butyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 140

Table 23. Original data of Figure 2c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each butyl ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 142

Table 24. Original data of Figure 3a and 3b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each propyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 144

Table 25. Original data of Figure 3c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each propyl ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 146

Table 26. Original data of Figure 4a and 4b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each ethyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 148

Table 27. Original data of Figure 4c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each ethyl ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 150

Table 28. Original data of Figure 5a and 5b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each 2-methylbutyl ester during
ripening and senescence. Each value is the average of ﬁve replications.
....................................................... 1 52

Table 29. Original data of Figure 5c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each 2-methylbutyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 153

 

Table 3

 

Table 33.
re
55

Table 34.

re
E;

Table 30. Original data of Figure 6a and 6b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each 2-methylpropyl ester during
ripening and senescence. Each value is the average of ﬁve replications.
....................................................... 154

Table 31. Original data of Figure 6c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each 2-methylpropyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 155

Table 32. Original data of Figure 7a and 7b (chapter 4). Absolute, corrected
GCIMS response (total ion counts) of each pentyl ester during ripening
and senescence. Each value is the average of ﬁve replications. . . . . 156

Table 33. Original data of Figure 7c (chapter 4). Fractions, corrected GCIMS
response (total ion counts) of each pentyl ester during ripening and
senescence. Each value is the average of ﬁve replications. ....... 157

Table 34. Original data of Figure 8 (chapter 4). Absolute, corrected GCIMS
response (total ion counts) of each acid during ripening and senescence.
Each value is the average of ﬁve replications. .................. 158

LIST OF FIGURES

CHAPTER 2

Figure 1. Possible biochemical pathways implicated in volatile ester formation in
apple fruit. ............................................... 35

CHAPTER 3

Figure 1. Representative gas chromatograph of the headspace of ‘Redchief
Delicious’ apples at climacteric. Most predominant ester peaks are
identiﬁed by numbers: 1. butyl acetate; 2. 2-methylbutyl acetate; 3. hexyl
acetate; 4. hexyl 2-methylbutanoate. .......................... 56

Figure 2. Ontogeny of total esters, ethylene and respiration (CO2 production)
during ripening and senescence of ‘Redchief Delicious’ apples. The
volatile proﬁle of apple fruit was tracked from 3 weeks prior to eight
weeks after the onset of the ethylene climacteric (indicated by dashed
vertical line). Each symbol represents the average of 5 replications. . . 57

Figure 3. Pattern of hexanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile profile was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total hexanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, 2-methylpropyl, butyl, 2-methylbutyl, pentyl, and hexyl esters of
hexanoic acid. (C) Ester proportions (% of total hexanoate esters). Each
symbol represents the average of 5 replications. ................ 59

Figure 4. Pattern of butanoate esters during ripening and senescence of
’Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total butanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, butyl, 2-methylbutyl, and hexyl esters of butanoic acid. (C) Ester
proportions (% of total butanoate esters). Each symbol represents the
average of 5 replications. ................................... 61

Figure 5. Pattern of propanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total propanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, butyl, 2-methylbutyl, and hexyl esters of propanoic acid. (C) Ester
proportions (% of total propanoate esters). Each symbol represents the
average of 5 replications. . . -. ................................ 63

xi

 

Figure 6. Pattern of acetate esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total acetate esters
(B) GCIMS response (TIC) of ethyl, propyl, 2-methylpropyl, butyl, 2-
methylbutyl, pentyl, and hexyl esters of acetic acid. (C) Ester proportions
(% of total acetate esters). Each symbol represents the average of 5
replications. ............................................. 65

Figure 7. Pattern of 2-methylbutanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total butanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, butyl, pentyl, and hexyl esters of 2-methylbutanoic acid. (C) Ester
proportions (% of total 2-methylbutanoate esters). Each symbol
represents the average of 5 replications. ....................... 67

Figure 8. Pattern of pentanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total pentanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl, and
propyl esters of pentanoic acid. (C) Ester proportions (% of total
pentanoate esters). Each symbol represents the average of 5
replications. ............................................. 69

Figure 9. Pattern of heptanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total heptanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, and butyl esters of heptanoic acid. (C) Ester proportions (% of
total heptanoate esters). Each symbol represents the average of 5
replications. ............................................. 71

Figure 10. Pattern of octanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total octanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, and butyl esters of octanoic acid. (C) Ester proportions (% of total
octanoate esters). Each symbol represents the average of 5
replications. ............................................. 73

Figure 11. Pattern of alcohols identiﬁed during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric which

xii

 

Figure ‘

D T f“ n\ N n\

CHAPTE

Figure 1.

 

 

corresponds to the harvest date (indicated by an arrow). Each symbol
represents the average of 5 replications. ....................... 74

Figure 12. Esters and alcohols content during ripening and senescence of
’Redchief Delicous’ apple fruit. (A) Concentration of 2-methylbutyl
acetate, butyl acetate, hexyl acetate, hexyl hexanoate and hexyl
butanoate esters. (B) Concentration of ethanol, butanol, 2-methylbutanol
and hexanol alcohols. (C) Same as (B) but without ethanol concentration.
Odor thresholds are indicated for 2-methylbutyl acetate, butyl acetate,
hexyl acetate, ethanol, butanol and hexanol respectively (Flath et al.,
1967). Each symbol represents the average of 5 replications. ....... 75

CHAPTER 4

Figure 1. Pattern of hexanol esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total hexanol esters
and ontogeny of ethylene. (B) GCIMS response (TIC) of acetic,
propanoic, butanoic, 2-methylbutanoic, and hexanoic esters of hexanol.
(C) Ester proportions (% of total hexanol esters). Each symbol represents
the average of 5 replications. ................................ 93

Figure 2. Pattern of butanol esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total butanol esters
and ontogeny of ethylene. (B) GCIMS response (TIC) of acetic,
propanoic, butanoic, 2-methylbutanoic, hexanoic, heptanoic, and
octanoic esters of butanol. (C) Ester proportions (% of total butanol
esters). Each symbol represents the average of 5 replications. ...... 95

Figure 3. Pattern of propanol esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total propanol esters
and ontogeny of ethylene. (B) GCIMS response (TIC) of acetic,
propanoic, butanoic, 2-methylbutanoic, pentanoic, hexanoic, heptanoic
and octanoic esters of hexanol. (C) Ester proportions (% of total propanol
esters). Each symbol represents the average of 5 replications. ...... 97

Figure 4. Pattern of ethyl esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total ethyl esters and
ontogeny of ethylene. (B) GCIMS response (TIC) of acetic, propanoic,
butanoic, 2-methylbutanoic, pentanoic, hexanoic, heptanoic and octanoic

xiii

esters of ethanol. (C) Ester proportions (% of total ethyl esters). Each
symbol represents the average of 5 replications. ................. 99

Figure 5. Pattern of 2-methylbutanol esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total 2-
methylbutanol esters and ontogeny of ethylene. (B) GCIMS response
(TIC) of acetic, propanoic, butanoic, and hexanoic esters of 2-
methylbutanol. (C) Ester proportions (% of total 2-methylbutanol esters).
Each symbol represents the average of 5 replications. ........... 101

Figure 6. Pattern of 2-methylpropanol esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total 2-
methylpropanol esters and ontogeny of ethylene. (B) GCIMS response
(TIC) of acetic, and hexanoic esters of 2-methylpropanol. (C) Ester
proportions (% of total 2-methylpropanol esters). Each symbol represents
the average of 5 replications. ............................... 103

Figure 7. Pattern of pentanol esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total pentanol esters
and ontogeny of ethylene. (B) GCIMS response (TIC) of acetic, 2-
methylbutanoic, and hexanoic esters of pentanol. (C) Ester proportions
(% of total pentanol esters). Each symbol represents the average of 5
replications. ............................................ 105

Figure 8. Pattern of acids identiﬁed during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric which corresponds
to the harvest date (indicated by an arrow). Each symbol represents the
average of 5 replications. .................................. 106

Figure 9. Pattern of hexanol, hexanoic acid and the associated ester (hexyl
hexanoate) during ripening and senescence for ‘Redchief Delicious’
apple fruit. Each symbol represents the average of 5 replications. . . 107

Figure 10.Pattern of ethanol, acetic acid and the associated ester (ethyl acetate)

during ripening and senescence for ‘Redchief Delicious’ apple fruit. Each
symbol represents the average of 5 replications. ............... 108

xiv

CHAPTER I: INTRODUCTION

 

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Consumers consider good quality fruits and vegetables to be those that
look good, are ﬁrm, and offer good flavor and nutritive value. They buy based on
appearance and feel, however their satisfaction and repeat purchases are
dependent upon good edible quality. The top three factors ranked by consumers
as most inﬂuencing their buying decisions, are ﬂavor, appearance and ripeness
(Kader, 2002).

Flavor perception is a process that links plant biochemistry with the
physiology and psychology of the consumer (Beaudry, 2000). Flavor is
composed of taste and aroma. While primarily the sugars, organic acids and
phenolics contribute to the fruit taste it is the production of speciﬁc organic
volatile compounds that determines our sense of aroma. The aroma is the
product of the interaction of volatiles molecules retro nasally with the nose
olfactory epithelium.

The olfactory system is the most sensitive of the ﬁve senses. It can detect
odors in parts per trillion, whereas receptors in the tongue can detect taste
compounds in parts per hundred (Baldwin, 2000). Aroma compounds contribute
heavily to the overall sensory quality of fruit and vegetables. lmportantly, the
aroma of some fresh horticultural crops including apples has received more
attention from both consumers and producers because they perceive insufﬁcient
aroma quality (Beaudry, 2000).

The aroma is a complex mixture of different volatile compounds whose
composition is speciﬁc to species and often to variety. There could be a
compound more typical for a speciﬁc fruit but in general, the overall aroma

quality is the sum of a large number of volatile compounds. In recent years, great

 

 

progres
analys'.

compo.

   

proport.

the olefir
TU” INUI
normally
all fruit I!
aplﬁles (
very div
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2001)

proﬁle ,
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preseP
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progress has been made due to advances in physicochemical methods of
analysis improving the isolation and identiﬁcation of a large number of volatile
compounds from plant aromas.

Although several of these aroma compounds are complex, large
proportions are relatively simple molecules which being volatile at physiological
temperatures account for fruit aroma. Paradoxically the most important, both
quantitatively and physiologically, volatile compound given off by ripe apples is
the oleﬁne, ethylene, which is not directly involved in the aroma or ﬂavor of the
fruit (Nursten, 1970). The aroma volatiles are usually present at very low levels,
normally in amounts of under a p.p.m. or even p.p.b (vlv). The volatile proﬁle of
all fruit is usually very complex. More than 300 volatiles have been isolated from
apples (Dimick and Hoskin, 1981). The nature of the volatiles involved is also
very diverse and includes esters, alcohols, acids, carbonyl compounds
(aldehydes and ketones), and many other chemical groups. The most abundant
on a weight basis are esters (78-92%) and alcohols (6-16%) (Dixon and Hewett,
2001)

Studies correlating consumer recognition of the produce with the volatile
proﬁle emanating from the produce have shown that only a small number of
compounds are responsible for consumer recognition of that commodity (VViIls et
al., 1998). In most fruit and vegetables, the characteristic aroma is due to the
presence of one or two compounds, which are termed “character impact
compound”. For apples, the key compounds claimed to be responsible for the
characteristic green aroma are hexanal and 2-hexenal, and for the ripe aroma

ethyl 2-methylbutyrate, 2-methylbutyl acetate, butyl acetate and hexyl acetate

 

 

(Plotto

 

mmea
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differert
compor

T
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(Plotto et al.,1999; Fellman, 2000). Ethyl 2-methylbutyrate is a minor component
of the aroma fraction but our olfactory senses are extremely sensitive. The
threshold concentration, or minimum concentration at which the odor of ethyl 2-
methylbutyrate can be detected organoleptically, was found to be 0.001 mL/L. At
different stages of maturation, different compounds become the dominant
component of ﬂavor.

The biosynthetic pathways for such a wide range of volatiles is also very
diverse. However, limited work has been done on elucidating the aroma
formation mechanisms. The biosynthesis is further complicated by the fact that
while some of these volatiles are synthesized in the intact fruit, others are
produced only when the fruit tissue is macerated (Knee, 1993). Volatile
precursors include amino acids, membrane lipids and carbohydrates (Figure 1).
As a preliminary study on aroma biochemistry the aim of this research was to
characterize the patterns in ester biosynthesis during ripening and senescence.
This information is hoped improve our understanding of the physiology and

biochemistry of ester formation in apples.

References

Baldwin, E. A., J. W. Scott, C.K. Shewmaker and W. Schuch 2000. Flavor trivia
and tomato aroma: biochemisty and possible mechanisms for control of
important aroma components. HortScience 35(6): 1013-1022.

. Beaudry, R. 2000. Aroma generation by horticultural products: what can we
control? Introduction to the workshop. HortScience 35(6): 1001-1002.

Dimick, P. S. and J. C. Hoskin. 1981. Review of apple ﬂavor - state of the art.
CRC Crit. Rev. Food Sci. and Nutr. 18(4): 387-409.

Dixon, J. and E.W. Hewett. 2001. Exposure to hypoxia conditions alters volatile
concentrations of apple cultivars. J. Sci. Food Agr. 81(1):22-29.

Fellman, J. K., T. W. Miller, and OS. Mattinson. 2000. Factors that inﬂuence
biosynthesis of volatiles ﬂavor compounds in apple fruit. HortScience
35(6): 1026-1033.

Kader , AA. 1992. Postharvest biology and technology: an overview, p.15-20.
In: A.A. Kader (ed.). Postharvest Technology of Horticultural Crops.
University of California, California.

Knee, M.. 1993. Pome fruits, p. 325-346. In: G.B.Se, J.E. Taylor, and GA.
Tucker (eds). Biochemistry of fruit ripening. Chapman & Hall, London.

Nursten, H. E. 1970. Volatile compounds: the aroma of fruits, p. 239-265. In:
Hulme A.C. (ed.). The biochemistry of fruits and their products. Food
Research Institute, NOI‘WICI'I, England, Vol.1, Academic Press. INC., New
York.

Plotto, A., MR. McDaniel, and JP. Mattheis. 1999. Characterization of ‘Gala’
apple aroma and ﬂavor: differences between controlled atmosphere and
air storage. J. Amer. Soc. Hort. Sci. 124:(4) 416-423.

Wills, R, B. McGIasson, D. Graham, and D. Joyce. 1998. Structure and
composition, p. 15-32. In: R., VVIIIS, B. McGIasson, D. Graham, and D.
Joyce (eds.). Postharvest. An introduction to the physiology & handling of
fruit, vegetables and ornamentals. University of New South Wales Press.
Ltda., Sydney, Australia.

CHAPTER II: LITERATURE REVIEW

Ar:
develop”
biogenet:
conditions
disruption

Ar:
Esters are
formed ma
saturated

lnte
using corrl
isolated 5i
nearly 30.:
all the vol;
acetate, 2.
malor con:
(Blackmar
2'Il'telhylb~
1996),

As,

P
_—

bUIId up IC
I
1977i The

a ’OW Conts

 

Aroma generation by apple fruit is spontaneous, relying primarily on the
developmental stage of the organ. Volatiles are produced by intracellular
biogenetic pathways inﬂuenced by genetic factors and by ripening and storage
conditions (Leahy and Roderick, 1999). Part of apple aroma is that due to tissue
disruption by chewing also (Dirinck et al., 1989).

Apple aroma depends upon a complex mixture of organic compounds.
Esters are quantitatively and qualitatively the most important compounds and are
formed mainly from 2- to 6-carbon alcohols and acids. They are usually
saturated and may include branched 4- and 5-carbon units.

Interest in apple volatiles began early in this century. Flath et al. (1967),
using commercial essence of Delicious apples as a source of apple volatiles,
isolated 56 volatiles compounds. Later Dimick and Hoskin (1981) reported that
nearly 300 volatiles have been isolated from apple of which 38% were esters. Of
all the volatile compounds identiﬁed, only a few esters: butyl acetate, hexyl
acetate, 2-methylbutyl acetate and ethyl-2methylbutanoate, are considered
major contributors to the characteristic apple-like aroma in most cultivars
(Brackmann et al., 1993; Fellman et al., 2000; Song and Bangerth, 1996). Hexyl
Z-methylbutanoate is reported also to be important in apple aroma (Rowan et al.,
1 996).

As an apple ripens naturally, the amount of low-bowling esters tend to
build up to a maximum after a period of several weeks (Williams and Knee,

1977). The best volatile composition in ‘Starkspur-Golden’ apples is comprised of
a low content of high boiling-point esters (butanoates) and alcohols and a high

content of low boiling-point esters like acetates (Vanoli et al., 1995). Among

them. PE'
aboider
‘Golden’ z
ethyl but?

methyl2~

 

 

 

 

(Panasiu-
1983).
Ot.
include: tr
hexanol.
methylbut
present in
crushing)
Hoskin, 1s
volatiles ti
Cu
commune.

ONE of ID E

APPLE Vo

AD
Co’nCIden
Aroma pri

them, pentyl and hexyl acetate were the most abundant esters in quantity. They
also identiﬁed 3-penten-2-ol considered as a typical compound of ripening
‘Golden’ apples. Overripeness in ‘Golden’ apples is correlated with the sum of
ethyl butanoate, ethyl propanoate, ethyl-2-methyl pr0panoate, methyl butanoate,
methyl-2-methyl butanoate, ethyl-2-methyl butanoate and ethyl pentanoate
(Panasiuk et al., 1980; Patterson et al., 1974; Vanoli et al., 1995; Willaert et al.,
1983). ’

Other volatiles considered key to apple ﬂavor from macerated apple
include: trans-2-hexenal, ethyl-2-methylbutanoate, ethyl butanoate, trans-2-
hexenol, hexyl acetate, acetate, b-damascenone, ethyl hexanoate and propyl 2-
methylbutanoate (Leahy and Roderick, 1999). However, some of them are not
present in fresh apples to a signiﬁcant degree, like trans-2-hexenal (formed upon
crushing) and b-damascenone (formed during heat processing) (Dimick and
Hoskin, 1981). Schwab and Schreier (1988) identiﬁed glycosidically bound
volatiles from Jonathan apple fruit.

Cultural and physiological factors affect the production of aroma
compounds of apple fruits (Brackmann et al., 1993) but fruit maturity is probably

one of the most signiﬁcant factors (Song and Bangerth, 1996).

APPLE VOLATILES DURING FRUIT DEVELOPMENT AND RIPENING

Apple fruit shows a large increase in 002 and ethylene production rates
coincident with ripening for what is classiﬁed as climacteric fruit (Kader, 1992).
Aroma production is closely linked to the onset of the ethylene climacteric and

continues to increase as ripening progresses. During ripening, there is a rapid

increase

 

(Fellman
Wiliaert e
ethylene

aroma vc
itis not c
concurre'

climacteri

 

 

 

 

Ea
state of th
which pea
1989; Ma
1973).

Ya
the tree.
Ionned a
3Utocata
”Perting
and 2~m
1993)_ 1
during n
produm-

DelieioL

increase in metabolites available for biosynthesis of the volatile molecules
(Fellman and Mattheis, 1995; Mattheis et al., 1991b; Romani and Ku, 1966;
VIﬂllaert et al., 1983; Williams and Knee, 1977). An increase in autocatalytic
ethylene production and respiratory activity may be essential for a characteristic
aroma volatile production (Fan et al., 1998; Song and Bangerth, 1996). However,
it is not clear whether the onset of biosynthesis of volatile compounds is
concurrent with, or precedes and perhaps plays a role in the initiation of, the
climacteric rise in fruit respiration (Fellman et al., 2000).

Early work trying to correlate production of volatiles with the physiological
state of the fruit indicates an increase in the production of volatile compounds,
which peaked just after the climacteric peak (Brown et al., 1966; Dirinck et al.,
1989; Mattheis et al., 1991b; Song and Bangerth, 1996; Tressl and Drawert,

1 973).

Yahia et al. (1990), analyzed apples during the maturation and ripening on
the tree. Most of the important odor-active volatiles (from apple juice) were
formed at or after the onset of ripening and their production followed the
autocatalytic evolution of internal ethylene. The ester concentration during
ripening of ‘Rome’ apples increased with advancing harvest date; butyl acetate
and 2-methyl-butyl acetate were the main compounds found (Fellman et al.,
1993). The acetate concentration of ‘Bisbee Delicious' apples also increased
during ripening with picking date (Mattheis et al., 1991b). The onset of volatile
production was delayed in early picked ‘Jonagold’ (Hansen et al., 1992), ‘Golden

Delicious’ (Dirinck et al., 1989) and ‘Starkspur-Golden’ (Vanoli et al., 1995)

apples 8'

apples. -

 

A;
synthesis
had a re:

fruit (Fan

requires r

     
 

biosynthe:

AVG and l

 

acetate 3.
initiation o‘
admn.
The
most Climg
associatio
EXplainec
with the TL
recognize!
deIEI'TT‘IITlir
OCCUI res L
ChIOTOpIaS
lipids an d
seq)nd 3T)

apples and the production was lower during ripening compared to later picked
apples.

Apples treated with aminoethoxyvinylglycine (AVG), that inhibits ethylene
synthesis, and with diazocyclopentadiene (DACP), that inhibits ethylene action,
had a reduced production of some volatile esters in both pre and postclimacteric
fruit (Fan et al., 1998). The authors suggested that biosynthesis of these esters
requires not only continuous ethylene action but also continuous ethylene
biosynthesis. On the other hand, acetate ester production was not affected by
AVG and DACP in postclimacteric fruit. The authors suggested that sufﬁcient
acetate and the enzyme(s) required for ester biosynthesis were present after the
initiation of apple ripening regardless of the status of ethylene production or
acﬁon. ’

The association of the climacteric with taste and aroma shifts holds for
most climacteric crops (Maul et al., 1998; Tressl and Drawert, 1973). This
association of the climacteric with maximum rates of ester formation was
explained by the fact that ester formation requires acyl-CoA, which is associated
with the fundamental metabolism of the cell (Nursten, 1970). Ethylene,
recognized as the ‘ripening hormone’ is responsible for the climacteric rise
determining the onset of the ripening of the fruit. Chemical and physical changes
occur resulting in changes in color, texture, and ﬂavor. For instance, the
chloroplast lamellae break down and the constituents of the membranes, both
lipids and proteins, are broken down and may be used as building materials for
secondary metabolites. The activity of the enzymes involved in these changes

increases during the climacteric producing fatty acids and amino acids, which

10

have be?

 

all fruits :
Tr :

with adva

 

changes ‘

(Guadag"

 

 

 

declined t
synthesis
are subse
Mattheis
Pr
which de
same pr
1991b).
disappe
appear
I“ ‘Gal

’nCrEa.

Chara
apple
and C
Dl’Opy

ethyr

have been shown to act as precursors of apple, banana and strawberry volatiles,
all fruits that produce esters as main aroma compounds.

The sequence of aroma volatile production in whole apple fruit changes
with advancing of ripeness. The aroma proﬁle in whole and crushed apple fruit
changes from aldehydes (“green-notes”) to esters (“fruity-notes) during ontogeny
(Guadagni et al., 1971; Mattheis et al., 1991b). The concentration of aldehydes
declined to no detectable levels by the end of the maturity, when the esters
synthesis start as ripening began. Apple fruit reduce aldehydes to alcohols that
are subsequently esten'ﬁed with carboxylic acids (Knee and Hatﬁeld, 1981;
Mattheis et al., 1991b).

Preclimacteric ‘Golden Delicious’ apples are rich in 02-06 aldehydes,
which decrease to trace amounts in climacteric fruits (Fellman et al., 2000). The
same progression was observed in ‘Bisbee Delicious’ apples (Mattheis et al.,
1991b). Likewise, there is in ‘Starkspur Golden’ apples a progressive
disappearance of aldehydes (hexanal and (E)-2-hexenal) and a gradual
appearance of acetate and butanoate esters during ripening (Vanoli et al., 1995).
In ‘Gala’, ‘Delicious’, ‘Rome’ and ‘Fuji’ apple fruit, acetate ester concentrations
increased during ripening as harvest maturity advanced (Fellman et al., 2000).

On the other hand, for some authors aldehydes are important to
characteristic apple aroma (Vanoli et al., 1995; Willaert et al., 1983). In McIntosh
apples ripe aroma was correlated with C-6 aldehydes (hexanal and 2-hexenal)
and overripeness was correlated with esters tentatively identiﬁed as ethyl
propionate, ethyl 2-methylpropionate, methyl butyrate, methyl-2-methylbutyrate,

ethyl butyrate, ethyl 2-methylbutyrate, and ethyl pentanoate (Panasiuk et al.,

11

1980). A
appies a
aromatic
produce:

(Vanoli e

of the vo

a.100=

UUU"

ESTER BIC

Di.r
takes plat
organizati
the fresh c
SUggeste.
PTOduces I
the ﬂesh g
SUbstrateS
activity (Fe
Coating frc

al.,1971)_

   
  

 

Ami:

1980). An increasing quantity of (E)-2-hexenal was found in ‘Starkspur Golden’
apples as the compound responsible for giving the aroma described as ‘ripe,
aromatic and fruity’ (Flath et al., 1967). Other point of view is that aldehydes are
produced during chewing (Flath et al., 1967) and could occur in ripen apples
(Vanoli et al., 1995), but during ripening they are ovenrvhelmed by the presence
of the volatile esters (Guadagni et al., 1971; Mattheis et al., 1991 b; Mattheis et

al,1995)

EerR BlOSYNTI-IESIS

Dirinck et al. (1989) reported that the generation of aroma esters in apples
takes place mainly in the peel, is oxygen-dependent and requires the
organization of intact tissue. Peel produced a greater quantity of volatiles than
the ﬂesh of intact fruit (Guadagni et al., 1971; VIﬁlliams and Knee, 1977). This
suggested that the primary biochemical system involved in aroma production
produces esters and that its activity is located principally in the skin rather than in
the ﬂesh of the fruit, apparently because of an abundance of fatty acid
substrates resulting from modiﬁed metabolic processes and enhanced enzymatic
activity (Fellman et al., 2000; Guadagni et al., 1971). Removing the oily, wax
coating from the skin did not reduce its ability to produce the esters (Guadagni et
al., 1971). Knee and Hatﬁeld (1981), on the other hand showed that the peel
has a more active esterifying system than the cortex but the system is
qualitatively similar in both tissues.

Amino acids, sugars and lipids all can act as precursors for ester

substrates. The ﬁnal reaction in the pathway for ester formation has been fairly

12

well cha

 

I

fermente
transfer"
destruct

I
CoA on t
functions

also beer

inhibitors

 

is a coen
activity (L

Th
between
The bran
methylpr.
al.. 1970
the Case
amino at
SUbSeqU
’0 2‘met
stralsht.
adds (F.
and DEX
by redUC

(Knee at

well characterized. It is better known in micro-organisms, in the process of
fermentation, in which two different enzymes are involved: alcohol acyl-CoA
transferase (AAT, EC 2.3.1.84) in the production of esters and esterase in the
destruction of esters. AAT catalyzes the transfer of an acyl moiety of an acyl-
CoA on to the corresponding alcohol (Dixon and Hewett, 2001), while esterase
functions mainly by hydrolyzing esters (Figure 1). Both enzymatic activities have
also been described in fruits (Sanz et al., 1997). Experiments using esterase
inhibitors have demonstrated that the esteriﬁcation of alcohols and acids in fruits
is a coenzyme A-dependent reaction and that esterase has only hydrolytic
activity (Ueda and Ogata, 1977 cited in Perez et al., 1996).

The carbon chain length of the alcohol portion of the esters varies
between 1 and 6 carbons. The carbon chain can be both straight and branched.
The branched-chains are believed to be derived from amino acids valine (2-
methylpropyI-), isoleucine (2-methylbutyI-), and leucine (3-methylbutyl—) (Myers et
al., 1970; Perez et al., 1992; Tressl and Drawert, 1973; Wyllie et al., 1995). In
the case of isoleucine, it has been proved that there is ﬁrst a deamination of the
amino acid forming 2-methylbutanoic acid, followed by decarboxylation and
subsequent reduction to 2-methylbutanol that competes with direct esteriﬁcation
to 2—methylbutanoate esters (Perez et al., 1992; Rowan et al., 1996). The
straight-chain alcohols are believed to be reduced forms of short-chain fatty
acids (Fellman et al., 2000; Knee and Hatﬁeld, 1981). Alcohols such as butanol
and hexanol are produced from fatty acids presumably by b-oxidation followed
by reduction in two stages from acetyl-CoA to aldehyde and aldehyde to alcohol

(Knee and Hatﬁeld, 1981). The reduction from aldehyde to alcohol is catalyzed

13

produc

througl

ahehyc
byhpox
the inter
synthesis
ability to
The form.
the tissue
the preser
or an 8Cyl-

Hexanol cl

 

resulting f:

 

by the action of alcohol dehydrogenase (ADH, EC 1.1.1.1) on aldehydes.
Treatments with aldehydes increased the content of all esters derived from the
corresponding alcohols, conﬁrming the activity of alcohol dehydrogenase in
apple (Bartley et al., 1985; De Pooter et al., 1983). Vanoli et al. (1995) found that
when acetaldehyde was low in concentration, esters reached their maximum
production. De Pooter et al. (1983) suggest that apples synthesize esters also
through aldehyde reduction.

The ubiquitous nature of ADH may result form the need to eliminate
aldehydes, which can be produced by fermentation during low oxigen stress and
by lipoxygenase activity following tissue disruption. Actually, ADH is involved in
the interconversion of alcohols and aldehydes to supply precursors for ester
synthesis and the production of other volatile compounds. Apple tissue has the
ability to metabolize added primary alcohols to acetate esters and aldehydes.
The formation of aldehydes implies the presence of alcohol dehydrogenase in
the tissue. It is also reported the formation of alcohols from acids which implies
the presence, in addition to alcohol dehydrogenase, of aldehyde dehydrogenase
or an acyl-CoA reductase (Knee and Hatﬁeld, 1981; Tressl and Drawert, 1973).
Hexanol could also be derived from hexanal or hexenal, which are fragments
resulting from the oxidative cleavage of linoleic or linolenic acids (Knee and
Hatﬁeld, 1981).

The acid portion of esters typically has a chain length from 2 to 8 carbons,
although there are exceptions. As for the alcohol portion of the molecule, both
straight and branched chains are common. The branched-chain compounds are

believed to be derived from the same amino acids as the alcohols: valine (-2-

14

methyl C
butanoa
derived ‘
SCFAS'
synthes ;
acids 08'
iMennec
ohdahor
esters wi‘

samml

 

 

Suggestt
Incorpora
d-1999}
fatty acid
SI’hthesis
esters, H
amvewl
as to the
enZYme I
Ibund in

fataboiiz
tmetha

methyl propanoate), isoleucine (-2-methyl butanoate), and leucine (-3-methyl
butanoate). The straight short-chain fatty acids (SSCFAs) are believed to be
derived from fatty acid metabolism. Two possibilities seem most likely: short
SCFAs may be result from the catabolism of previously formed fatty acids or
synthesized de novo. Bartley et al. (1985) supports the idea that long chain fatty
acids can be precursors to straight chain alcohols, aldehydes and acids, all
intermediates for ester formation. They concluded from their results that
oxidation of fatty acids is the likely source of precursors for the synthesis of
esters with alkyl group (Ch-2, Cn-4) and that the precursors arise because there
is a rate limiting step in the b-oxidation pathway. Precursor feeding studies
suggest that b-oxidation is responsible for the synthesis of the SCFAs
incorporated into esters (Bartley et al., 1985; Brackmann et al., 1993; Rowan et
al., 1999). The other possibility is that SCFAs are derived from the pathway of
fatty acid synthesis (Tan and Bangerth, 2001). Nursten (1970) implied lipid
synthesis as the source of even numbered carbon chains that eventually form
esters. He suggested that the intermediates acyl-ACP of fatty acid biosynthesis
are very likely to be susceptible to alcoholysis to the corresponding ester as well
as to the normal hydrolysis to the free acid. However, the thioesterase B, the
enzyme which catalyses the release of SCFAs from the synthetase, has been
found in only a few specialized mammalian organs. Fatty acids can be also
catabolized through the lipoxygenase pathway. However, this is most active in
fruits that produce volatiles by disruption of cells (Bartley et al., 1985; Rowan et

al., 1999).

15

l<

apple tiS
diffusior‘
ck of :-
rates. Fe
their cor:
A:

readily in

 

 

 

 

productic
Knee an:
These ex
primary I
Effects 0
strawber
the later
”Wipe fr
the imm
DTeCUrS(
Working
AAT ax
when n.
Yamasl
’BSPEE

acetate

Knee and Hatﬁeld (1981) suggest that the levels of esters and alcohols in
apple tissue result from an equilibrium between synthesis, hydrolysis and
diffusion from the tissue. Thus, low concentration of esters could be caused by a
lack of precursors, low esterifying activity, high esterase activity or high diffusion
rates. Fellman et al. (2000) suggest that the balance between acetate esters and
their corresponding alcohols may be regulated by esterase activity.

Applied vapors or solutions of alcohols, organic acids and aldehydes are
readily incorporated into esters in intact fruit tissues with low or no ester
production (Bartley et al., 1985; Berger and Drawert, 1984; Fomey et al., 2000;
Knee and Hatﬁeld, 1981; Williams and Knee, 1977; Wyllie and Fellman, 2000).
These experiments support the hypothesis that substrate availability is the
primary limitation in the production of esters, having qualitative and quantitative
effects on the volatile esters proﬁle. Yamashita et al. (1977), working with
strawberry, showed that the ester-forming enzyme activity is induced only during
the later stages of maturation in strawberry fruit, since no activity was found in
unripe fruits. Therefore, they concluded that the lack of most of the volatiles in
the immature strawberry fruit is probably due to the absence of volatile
precursors and the enzyme forming systems. However, Perez et al. (1993),
working with strawberry found that the high level of esterase activity difﬁcult the
AAT extraction. This would explain why no ester formation has been detected
when homogenized strawberry tissue was incubated with different alcohols by
Yamashita et al. (1977). Furthermore, Mattheis et al. (1991b) working with intact
‘Bisbee Delicous’ apple fruit detected some esters, most notably 2-methylbutyl

acetate and butyl acetate, before the onset of ethylene production. Unripe peel

16

and cor?
their cor
that the t
maturity
ester for:
at al.. 19
preclima:
substrate
before the
experime
substrate.

aroma bic

 

 

AA
of AAT prr
the alcohc
2000; Olia
AAT was (
Sistem in
StraWbern
aCyl dOn Or
BI al., 199;
With Straig
Garbo” ”Ur

acted on v

and cortex tissue were capable of esterifying butanol and 2-methyl propanol to
their corresponding acetates (Knee and Hatﬁeld, 1981). These results suggest
that the enzymes needed for ester synthesis are functional prior to physiological
maturity, indicating that alcohol substrate availability may be the limiting factor in
ester formation. Precursor feeding studies in preclimacteric apple fruit (De Pooter
et al., 1983; Knee and Hatﬁeld, 1981; Song and Bangerth, 1994) and in
preclimacteric banana fruit (Jayanty et al., 2002) demonstrated that the supply of
substrates seems to be the limiting factor, rather than the amount of AAT present
before the onset of ripening. Rowan et al. (1998) suggested from their feeding
experiments with amino acid precursors that there may be competition between
substrates, and that enzymatic activity as well as substrate availability may limit
aroma biosynthesis.

AAT speciﬁcity also plays a key role in this process. Different isoenzymes
of AAT present in different fruits have different preferences for the acyl-CoA and
the alcohols. This preference is reﬂected in the volatile proﬁle (Fomey et al.,
2000; Olias, et al., 1993, 1995; Perez et al., 1993; Ueda et al., 1992). Before
AAT was characterized, Knee and Hatﬁeld (1981) suggested that the esterifying
system in apple has a relative speciﬁcity for longer carbon chain alcohols.
Strawberry AAT was found to prefer hexanol when acetyl-CoA was used as an
acyl donor although methanol and ethanol were not tested as substrates (Perez
et al., 1993). Moreover, the strawberry AAT enzyme seemed to be more active
with straight-chain alcohols than against branched-chain alcohols of the same
carbon number. Although it had slightly greater activity with acetyI-CoA, AAT

acted on various acyl-CoAs (propionate and butanoate). Differences exist among

17

 

specific:
Delicious
certain s-
(De Poo:
apple ar:
identity.

Tr.

etal.,195

 

enzl’rt'ie v
1985). St
ternDeratL
1993Iltv
et all, 199
20003) ar
2000b)_ T
ESTs hav
Arab/Corps

acyl-CoA and alcohol speciﬁcities between strawberry and banana AAT enzymes
(Olias et al., 1995). In both cases, there was a clear correlation between
substrate speciﬁcity and volatile esters present in the aroma of each fruit (Perez
etaL,1992)

Fellman et al. (2000) suggest that apple AAT probably exhibits substrate
speciﬁcity similar to the ﬁnal products of the reaction. Treatments of Golden
Delicious apples with aldehydes and carboxylic acids suggest that there is a
certain selectivity of the apple AAT in the use of the carboxylic acid precursors
(De Pooter et al., 1983). They supported the hypothesis that the composition of
apple aroma is determined by not only the availability of acids but also by their
identity.

The enzyme AAT has been puriﬁed and characterized in banana (Harada
et al., 1985) and in strawberry (Perez et al., 1993; Perez et al., 1996). The AAT
enzyme was localized in the soluble fraction of banana pulp cells (Harada et al.,
1985). Strawberry AAT showed to have a pH optimum of 8.0 and optimum
temperature of 35°C and an apparent molecular mass of 70 kDa (Perez et al.,
1993). It was suggested that AAT could be a membrane-bound enzyme (Perez
et al., 1996). Two AAT genes have been cloned from strawberry (Aharoni et al.,
2000a) and one gene has been identiﬁed in banana and apple (Aharoni et al.,
2000b). The size of the AAT gene family in these crops is not known, but several
ESTs having high sequence similarity to AAT have been found in the
Arabidopsis genome (Mekhedov, personal communication). AAT enzymes

appear to be a very heterogeneous group with a few common characteristics.

18

here;

 

(Sanz e

F
ldusua
eahsm
aomyc
maximu
vanehes
pmmmr
showed
Suggest

subseq:

pubhsh
CHAVAT
FeHnna-
usedir

dUﬁng

StageS
aroma
onsetc
Sifavvb.

mace r.

The relationship between AAT and lipid metabolism in fruits remains unknown
(Sanz et al., 1997).

Perez et al. (1996) studied the AAT activity proﬁle during maturation of
four strawberry varieties. Only in one variety, AAT activity was detected at the
early stages of maturity and all varieties showed an increase in AAT speciﬁc
activity during maturation. Both absolute and speciﬁc AAT activities reached a
maximum and then a clear decrease at the overripe stage. Differences among
varieties were found not only in relation to maximum AAT values but also in the
pattern of AAT activity during fruit maturation. However, the AAT speciﬁcity
showed similar results as previously reported (Perez et al., 1993). They
suggested that high AAT activity should result in higher ester production and
subsequently in fruits with enhanced aroma.

No study on apple AAT changes during fruit development has been
published; only preliminary studies on the effect of different storage conditions
on AAT activity have been carried out (Fellman et al., 1991;Fellman et al., 1993;
Fellman and Mattheis, 1995; Ke et al., 1994). Non-treated ‘Rorne’ apple fruit
used in Fellman and Mattheis (1995) study showed an increase in AAT activity
during the climacteric.

Jayanty et al. (2002) detected AAT gene expression in banana fruit of all
stages of ripening. The mRNA for AAT began to accumulate before the onset of
aroma production and the maximum level of expression was detected at the
onset of natural ester biosynthesis. Similar results were found in white
strawberry where AAT expression increased as ripening and color change took

place (Aharoni et al., 2000a).

19

of volat

 

unsaturs
presume
hexana'|
chain es

unsatura

hexenal.

 

1999). F.
apple wt
increase
chloropl
lipoxyge

and trar

PREHAF

I
Impact
Tole in
Strains
80me

FOFHE

egter

The enzyme lipoxygenase may play a role in determining the composition
of volatile compounds in apple (De Pooter et al., 1983; Fellman et al., 2000). The
unsaturated fatty acids linoleic (C18:2 D9”) and linolenic (C18:3 09"”) were
presumed to be precursors of the carbonyl compounds like the aldehydes
hexanal and cis-3 hexenal (T ressl and Drawert, 1973). Unsaturated straight-
chain ester volatiles may also be produced by the action of lipoxygenase on
unsaturated fatty acids through the intemnediacy of the C-6 aldehydes, 32-
hexenal, 2E-hexenal, and hexanal by the lipoxygenase pathway (Rowan et al.,
1999). Fellman et al. (2000) cites a work from Pillard (1986) in ‘Golden Delicious’
apple where they associated the degreening occurred during ripening with an
increase in membrane galactolipids rich in linolenic and linoleic acids from
chloroplast degradation. They suggested that these lipids are oxidized by
lipoxygenase activity and/or 13.-oxidation generating the C6 aldehydes hexanal

and trans 2-hexenal.

PREHARVEsr FACTORS AFFECTING AROMA BlOSYNTl-IESIS

Many preharvest factors can affect the development of fruit aroma by
impacting ester biosynthesis. Cultivar and rootstock genotype have an important
role in determining the ﬂavor quality. Genetic differences between ‘Delicious’
strains can alter the ﬂavor pattern in apple ﬂesh. However, there appears to be
some similarity in the major esters (Fellman et al., 2000). In ripe strawberries,
Fomey et al. (2000) found both quantitative and qualitative differences in the

ester volatiles evolved from different cultivars.

20

 

whethe
compos
opooshi
reducti:

levels 01

 

pigment.
synthes
synthes
found tr

of AAT.

Tespon
hfgh in
DhOSpt
COT‘itup

__A
(o
(J)
(I)
y

Sigmﬁ
Delici.
the a\
Coy-np

acids

Miller et al. (1998) conducted studies in ‘Delicious’ apples to examine
whether there is a relationship between red coloration and ﬂavor volatile
composition. The effect of canopy position on acetate ester production is
opposite to that on anthocyanins, suggesting that the trade-off for high color is a
reduction in ﬂavor volatile concentrations. Fellman et al. (2000) detected lower
levels of butyl acetate and hexyl acetate in apples with higher proportion of
pigmented skin cells. They explained this reduced capacity of acetate ester
synthesis by substrate availability limitation. The acetate moieties are used in the
synthesis of anthocyanins molecules deposited in peel cell vacuoles. They also
found that higher coloring mutations of ‘Delicious’ had lower levels of the activity
of AAT.

Nutrient balance is important for normal production of the compounds
responsible for taste and aroma. Esters from freshly-harvest apples from trees
high in phosphorus had a higher ester production than those from trees low in
phosphorus (Brown et al., 1968). Nitrogenous fertilizers, when used in
conjunction with potassium and phosphorus, increased the amount of volatile
compounds produced by apples (Somogyi et al., 1964 cited in Brown et al.,
1968). However, more recently Fellman et al. (2000) found no statistically
signiﬁcant effect of nitrogen nutrition on the volatiles proﬁle of ‘Redspur
Delicious’ apples. The authors did not ﬁnd an effect of nitrogen application on
the availability of amino acid related precursors. Degradation of chloroplast
components and associated macromolecules may create a large pool of amino

acids residues needed for synthesis of branched-chain esters.

21

apple C.

and w'.?

 

ammal

biosynt:

POSTHI

alcohc
state c
emiss
hawe'

Fena

Ferrandino et al. (2001) reported the effect of environmental conditions on
apple quality and on aroma production. Fruit from higher altitudes (1000 m a.s.l.)
and with north exposure had higher quantities of alcohols responsible for fruit
aroma. Little is known regarding the impact of other cultural practices on volatile

biosynthesis (Fellman et al., 2000).

POSTHARVEST FACTORS AFFECTING AROMA BIOSYNTHESIS

Harvest date and storage regime can positively or negatively effect esters,
alcohols, and hydrocarbons (Girard and Lau, 1995). There is an effect of the
state of maturity of fruit prior to being placed in store, with a greater volatile
emission from fruit from latter harvests (climacteric stages) than from earlier
harvests (preclimacteric stages) (Bangerth et al., 1998; Brachmann et al., 1993;
Ferrandino et al., 2001; Mattheis et al., 1995; Williams and Knee, 1977).

Controlled atmosphere (CA) storage is commonly used to delay ripening
and extend the storage life of apples (Fellman et al., 1993). CA storage utilizes
oxygen and carbon dioxide concentrations of about 1 to 5 percent for each gas
(Kader, 1992). Many investigations have revealed that CA storage signiﬁcantly
suppresses aroma production (Brackmann et al., 1993; Fellman, et al., 2000;
Girard and Lau, 1995; Ke et al., 1994; Mattheis, et al., 1995, 1998; Tough and
Hewett, 2001 ). However the last steps of the ester biosynthesis pathway are
active after fruit is removed from CA (Bartley et al., 1985; Brackmann et al.,
1993; Knee and Hatﬁeld, 1981). Investigations in the response of apple AAT to

regular air and CA storage suggest that inhibition of ripening-related events

22

hﬁuenc

 

and M?

C

I

which 0:1
O. and:

Song e:

as buty?

 

reduced
were ide
foundin
conditior

eiirninat

decrees
esters 5
et al., 1
re'iated
pTOducj
the tilt;

GGTIVed

aCEtald.
(Manhe

Volatile

inﬂuences subsequent AAT activity after storage (Fellman et al., 1993; Fellman
and Mattheis 1995; Fellman et al., 2000).

Other postharvest technique is modiﬁed atmosphere packaging (MAP)
which objective is to generate an atmosphere similar to CA, with sufﬁciently low
02 and/or CO2 to inﬂuence the metabolism of the products being packaged.
Song et al. (1997) demonstrated that the impact aroma character volatiles, such
as butyl acetate, hexyl acetate, and 2-methylbutyl acetate were signiﬁcantly
reduced under the low 02 conditions. On the contrary, ethanol and ethyl acetates
were identiﬁed as the major volatile compounds. The increased AAT activity
found in strawberries under passive modiﬁed atmosphere (MA) storage
conditions (CO2 > 30%) could be attributed to a detoxifying function of AAT to
eliminate the excess of ethanol generated by fermentation (Perez et al., 1996).

Ultralow oxygen (ULO) storage conditions in ‘Golden Delicious’ apples
decreased straight-chain esters such as butyl acetate, while branched-chain
esters such as 2-methylbutyl acetate were suppressed by high CO2 (Brackmann
et al., 1993). Suppression of aroma production by ULO conditions seems to be
related to low fatty acid synthesis and/or degradation. Suppression of aroma
production under high CO2 concentrations seems to be related to an inhibition of
the tricarboxylic acid (T CA) cycle from which most amino acid precursors are
derived (Brackmann et al., 1993).

Fermentation induced by anaerobiosis produces large quantities of
acetaldehyde and ethanol, which increases the production of ethyl esters
(Mattheis et al., 1991a). Brief period of hypoxic conditions (100% C02) alters

volatile proﬁle of apple fruit (Dixon and Hewett, 2001; Fomey et al., 2000). The

23

authors
compe:
andlor '

biosyn'.

 

C|
pyruva:
accumL

concen‘.

reaction

 

Enhanc.
vapors i
that the
compe:

I
MCPy l.
Pemeiv
apple f
of bios
CODtrot
QTEen .

C0”ch

authors suggested that this enhancement of ethyl esters might be due to
competitive inhibition by ethanol of biosynthesis of esters from other alcohols
and/or to a change in AAT activity and/or substrate speciﬁcity of the volatile
biosynthetic pathway.

CA treatment in strawberry enhanced activities of fermentation enzymes
pyruvate decarboxylase and alcohol dehydrogenase causing ethanol
accumulation. As the AAT activity was slightly decreased, the increased ethanol
concentration competes with other alcohols for carboxyl groups for esteriﬁcation
reactions and the biosynthesis of ethyl esters increase (Ke et al., 1994).
Enhanced apple sensory quality upon application of high amounts of ethanol
vapors decreased the concentration of some butyl- and hexyl esters indicating
that the esteriﬁcation of acyl moieties (especially C4 and longer) is likely a
competitive reaction (Berger and Drawert, 1984).

Apples treated with the new growth regulator 1-methylcyclopropene (1-
MCP), that prevents the action of ethylene have longer storability and are
perceived to be less ripe. It decreases the biosynthesis of aroma volatiles by
apple fruit to levels similar to those of fruit given CA storage and delay the onset
of biosynthesis (Ferenczi and Beaudry not published), yet more acceptable than
control apples (Lurie et al., 2002). Similarly, application of 1-MCP on mature
green bananas caused a quantitative but not a qualitative change in the
composition of the aroma volatiles (Golding et al., 1999).

Dimick and Hoskin (1981) cited works that studied the effect of water loss
on ﬂavor volatiles. The measured quantity of esters increased while the alcohols

decreased when the rate of weight loss per week increased.

24

MEASU1

concef‘
distinct
can far
manor

for thos

 

solvent
direct h
2090)‘
Passing
such a:
0r GC/i

diSIr‘itEi

fruit tis

MEASURING THE VOLATILEs COMPONENTS OF APPLE FRUITS

Human olfaction is exceptionally sensitive, capable of detecting very low
concentration of volatiles compounds. Humans can discriminate over 10,000
distinct odors. Gas chromatography (GC) detectors vary in sensitivity and they
can far exceed the human nose in sensitivity with compounds with little or no
olfactory effect, but they can be up to 10,000 times less efﬁcient than the nose,
for those compounds which the nose most readily senses (Nursten, 1970).

Certainly when considering apple volatiles, the primary method of
component separation is GC and although many identiﬁcation methods exist, the
most useful is GC-MS (Dimick and Hoskin, 1981). Earlier volatiles analyses have
been done by the classical ﬂavor isolation procedures of steam distillation and/or
solvent extraction. More recently, investigators have employed basically either
direct headspace or dynamic headspace purge-and-trap methods (Baldwin et al.,
2000). The purge-and-trap method collects the volatile compounds from the air
passing over the whole fruit trapping and concentrating them on a solid support
such as charcoal or Tenax. The trap is later heated to release volatiles into GC
or GCIMS systems. Aroma volatile analysis can also be by extraction of
disintegrated tissue or direct measurement of the volatiles in the headspace of
fruit tissue discs (Knee and Hatﬁeld, 1976).

However, these methods are expensive and time-consuming processes.
The newest method used is solid phase microextraction (SPME), a rapid
sampling technique where volatiles interact with a ﬁber-coated probe that is

inserted into the headspace of a sample and then transferred to GCIMS injection

25

port where the volatiles are desorbed (Matich et al.,1996; Song et al., 1997;
Song et al., 1998).

Aside from GC and GCIMS methods, there are sensor arrays called
‘electronic noses’ (EN) that are useful for discriminating one sample from
another based on the volatile proﬁle, rather than for identiﬁcation/quantiﬁcation
(Baldwin et al., 2000). An EN is comprised of a series of nonspeciﬁc gas
sensors that are useful for aroma discrimination since their electrical resistance
properties are altered by the adsorption of volatile compounds produced by the
sample (Maul et al., 1998).

To know which aroma compounds are contributing to ﬂavor, aroma
extraction dilution analysis (AEDA) or “Charm” analysis use a sniff port on a GC
while diluting the sample. A simpler method is to establish odor thresholds (the
level at which a compound can be detected by smell). This is done in the food or
in some similar medium since odorants’ volatility can change with polarity and
viscosity. Log odor units can then be calculated from the ratio of the
concentration of a component in a food to its odor threshold. Volatile compounds
with positive odor units are assumed to contribute to the ﬂavor of a food, while
those with negative units may not (Baldwin et al., 2000).

The concentration of the volatiles in air passing over apples depends on
the permeability of the tissue, the concentration of the volatiles in the peel and/or
cortex and the extent of enzyme hydrolysis of esters passing through the peel.
According to Knee and Hatﬁeld (1976) experiment, the complexity of factors
inﬂuencing the composition and quantities of volatiles compounds released by

whole apples precludes general conclusions about their relation to internal

26

concentrations, and the evaluation of their role in apple ﬂavor. Low ﬂow rates of
air passing over apples would cause an accumulation of esters over several
days, while fast ﬂow rates would cause a similarly slow decline. Thus, it is
erroneous to calculate rates of production from concentrations of esters found in
air streams passing over apples (Knee and Hatﬁeld, 1976).

Each combination of techniques results in a slightly different volatile
proﬁle. Methods used to collect and analyze volatiles can cause the loss of
certain compounds. In our case, analysis of headspace compounds by SPME is
dependent on their individual vapor pressure and their afﬁnity for the ﬁber. The
more volatile compounds are present in higher concentrations in the chamber
headspace. This reﬂects the compound’s contribution to the fruit aroma but does
not give its true concentration in the tissue. In addition, it has been demonstrated
that the less volatile high molecular weight aroma compounds evaporate slowly
form the surface of the apples and are depleted from the headspace because of
very rapid adsorption by the SPME ﬁber (Matich et al., 1996). Disruption of the
fruit through homogenization removes barriers to diffusion and allows for the
determination of true concentrations, but causes enzymatic changes in the
volatile proﬁle especially the production of lipoxygenase products such as the

aldehydes hexanal, hexanal and their alcohols (Fomey et al., 2000).

27

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2373.

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Olias, J. M., C. Sanz, J.J. Rios, and AG. Perez. 1995. Substrate speciﬁcity of
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Patterson, B.D., S.G.S. Hatﬁeld, and M. Knee. 1974. Residual effects of
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Perez, A.G., J.J. Rios, C. Sanz, and J.M.Olias. 1992. Aroma components and
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Perez, A. G., C. Sanz, and J.M. Olias. 1993. Partial puriﬁcation and some
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Sanz, C., J. Olias, and AG. Perez. 1997. Aroma biochemistry of fruits and
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Phytochemistry of Fruit and Vegetables. Oxford University Press Inc.,
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Technol. 6(3-4):225-234.

33

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Wyllie, S. G. and J. K. Fellman. 2000. Formation of volatile branched chain
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3496.

Yahia, E.M., T.E. Acree, and PW. Liu. 1990. The evolution of some odour-active
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Yamashita, |., K. Lino, Y. Nemoto, and S.Yoshikawa. 1977. Studies on ﬂavor

development in strawberries. 4. Biosynthesis of volatile alcohol and esters
from aldehyde during ripening. J. Agric. Food Chem. 25(5):1165-1168.

34

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35

CHAPTER III:

PATTERNS IN THE ALCOHOL PORTION OF ESTERS PRODUCED DURING RIPENING AND
SENESCENCE OF ‘REDCHIEF DELICIous’ APPLE FRUIT.

36

INTRODUCTION

Aroma compounds contribute signiﬁcantly to the ﬂavor of all fresh fruits.
However, the aroma of some fresh crops including apples has received more
attention from both consumers and producers because they perceive insufﬁcient
aroma quality (Beaudry, 2000).

There are several classes of compounds that Contribute to aroma. Esters
comprise a broadly distributed class of aroma volatiles among various fruit
species and contribute signiﬁcantly to the aroma of apple (Ma/us x domestica
Borkh.), pear (Pyrus communis), melon (Cucumis melo), banana (Musa sp), and
strawberry (Fragan'a x ananassa Duch.) fruit. Many factors can affect the
development of fruit aroma by impacting the ester biosynthesis: cultivar, growing
conditions, fruit maturity, and also storage conditions (Fellman, 2000).

Esters are formed from fatty acids and alcohols (Figure 1 in Chapter 2).
The enzyme alcohol acyl-COA transferase (AAT, EC2.3.1.84) catalyzes the union
of an alcohol and the acyl-CoA derivative of a fatty acid. Substrate availability is
the primary limitation in the production of esters after storage, having qualitative
and quantitative effects on the volatile ester proﬁle (Knee and Hatﬁeld, 1981;
Wyllie and Fellman, 2000). Furthermore, AAT has speciﬁc preferences for acyl-
CoAs and alcohols, which tends to be reﬂected in the volatile proﬁle (Olias et al.,
1995; Perez et al., 1993). It is not known how many different AAT isozymes
could be present in apples; only one AAT gene has been identiﬁed. No study on
apple AAT activity during fruit development has been published; only preliminary
studies on the effect of different storage conditions on AAT activity has been

carried out (Fellman et al., 1993; Fellman and Mattheis, 1995; Ke et al., 1994).

37

The carbon chain length of the alcohol portion of the ester varies between 2 and
6 carbons and the acid portion has typically a chain length from 2 to 8 carbons.
The carbon Chains of the alcohols or fatty acids can be straight or branched. The
branched-chain compounds are believed to be derived from amino acids
(Nursten, 1970; Perez et al., 1992; Tressl and Drawert, 1973; Wyllie et al.,
1995). The straight short-chain fatty acids (SSCFA), between 2 and 8 carbon
length, are believed to be derived from fatty acid metabolism, either degradation
(Bartley et al., 1985; Brackmann et al., 1993; Fellman et al., 2000; Nursten,
1970; Rowan et al., 1996, 1999) or synthesis (Tan and Bangerth, 2001). The
relationship between AAT and lipid metabolism in fruit remains unknown.
Although considerable progress has been made in isolating and identifying a
large number of volatile compounds from plant aromas, less work has been done
on elucidating the aroma formation mechanism.

The aim of this research was to Characterize the patterns in ester
biosynthesis during ripening and senescence of ‘Redchief Delicious’ apple to
better understand the biochemical origin and fate of these organoleptically
signiﬁcant compounds. Apple fruit were tracked throughout ripening and selected
fruit for analysis based on internal ethylene levels. At each stage evaluated,
respiration and ester production was measured for ﬁve representative fruit.
Developmentally dependent patterns in esters were evaluated. An ester matrix
was established based on precursor acids and alcohols (Table 1). One axis of
the matrix included alcohols (ethanol, propanol, 2-methylpropanol, butanol, 2-
methylbutanol, pentanol, and hexanol) and the other axis acids (acetate,

propanoate, butanoate, 2-methylbutanoate, pentanoate, hexanoate, heptanoate,

38

and octanoate). Few alcohol/acid combinations were not detectable. In this
paper, we focused on patterns evident in the alcohol portion of esters classed by

the acid moiety during ripening and senescence.

MATERIALS AND METHODS

‘Redchief Delicious’ apples [Malus sylvestn’s (L) Mill. var. domestica
(Borkh) Mansf.] were harvested every three to four days at the Michigan State
University Horticultural Teaching and Research Center, East Lansing, MI,
beginning three weeks prior to the onset of the climacteric and continuing until
fruit were considered to have initiated ripening based on internal ethylene
content (IEC). The beginning of the climacteric rise was considered to occur
when the internal ethylene content was about 0.2 uUL. The harvest date
occurred on October 3‘“, which was day 25 of the experiment. Distinct patterns in
the ester production were evident.

After the initiation of ripening, the remaining fruit were harvested and held
at room temperature for analysis continuing fruit selection for 45 days. Thus, 18
different stages of development of ‘Redchief Delicious’ apple fruit ranging from
unripe through senescent over a period of 70 days were measured. The average
IEC of twenty representative fruits at each stage was determined and those ﬁve
fruit nearest the average were Chosen for ester evaluation.

The IEC was determined by withdrawing a 1-mL gas sample from the
interior of apples and subjecting the gas sample to gas Chromatographic (GC)
analysis. The gas chromatograph (Carle Series 400 AGC; Hach Co., Loveland,

Colo.) was ﬁtted with a 6-m-Iong, 2-mm-i.d. stainless-steel column packed with

39

activated alumina and detection was via a ﬂame ionization detector. The
ethylene detection limit was approximately 0.005 uL.L“. Ethylene concentrations
were calculated relative to a certiﬁed standard (Matheson Gas Products,
Chicago, III.) with an ethylene concentration of 0.979 LILL“.

Volatile analysis procedure was done as described by Song et al.
(1997,1998). Ester emissions were sampled by sealing one fruit in each one of
ﬁve 1-liter Teﬂon TM chamber. In order to reach a steady-state concentration of
apple fruit volatiles in the headspace over the apples, the fruit were maintained
in the chambers for approximately three hours at 22°C and the chambers were
ventilated with pure air at a rate of approximately 30 mUmin. One chamber with
no fruit was used as a blank.

A 1-cm long solid-phase microextraction (SPME) ﬁber coated with a ﬁlm
thickness of 65 pm of polydimethylsiloxane/divinylbenzene (Supelco Co.,
Bellefonte, PA) was used to adsorb the volatile sample. The SPME ﬁber was
preconditioned by baking overnight at 260'C.

The ﬁber was manually inserted through a Teﬂon-lined half-hole septum
into a glass ‘tee’ located at the outlet of the Chambers. Once in the glass ‘tee’
outlet, the ﬁber was extended to absorb volatiles for ﬁve minutes. The ﬁber was
then retracted prior to removal from the sample container.

Ester analysis was by GCItime-of-ﬂight mass spectrometry (MS). The SPME
ﬁber was inserted in the glass-lined, splitless injection inlet of the GC (230°C)
and desorbed for 5 minutes. The volatiles were cryofocussed oncolumn using a

liquid nitrogen cryo trap.

40

The desorbed ﬂavor compounds were separated by a Hewlett-Packard
6890 GC with a capillary column (Supelcowax, 15 m X 0.1 mm i.d., 0.25 pm
coating ﬁlm) (Supelco Co. Bellefonte, PA). The temperature of the GC was
programmed from 40 to 240 °C at 50 'Clmin. A constant mass ﬂow rate (0.5
mL/min) of the carrier gas (He) in the column throughout the run was maintained.
The identiﬁcation and quantiﬁcation of the volatiles were by comparison with the
National Institute of Standards and Technology database and authenticated
standards. Quantiﬁcation for selected compounds was accomplished using gas
standards. Gas standards were created from a mixture of equal volumes of the
neat oils of 13 compounds. A sample of 0.5 uL was taken by using a Hamilton
1.0 uL syringe, which was discharged onto a ﬁlter paper disk. The ﬁlter paper
was immediately dropped into a 4.4-L glass volumetric ﬂask ﬁtted with a ground-
glass stopper containing a gas-tight Mininert valve (Alltech Assoc, Inc.,
Deerﬁeld, IL). A new standard was made every month. Volatile aroma
compounds were purchased from Sigma Co. and Fluka Chemical Corp. The
compounds included in the standard were: 1-butanol, 1-hexanol, cis-3-hexen-1-
ol, ethyl alcohol, acetaldehyde, 1-methyI-1-butanol, n-butyl acetate, hexyl
acetate, hexyl butyrate, hexyl hexanoate, 3-methylbutyl acetate, 2-methylbutyl
acetate, and famesene mixture. For all compounds identiﬁed, not all standards
were available.

For each sample, all target compounds were identiﬁed. The peak area
was determined under the unique ion ID for each speciﬁc compound and the
total ion count (TIC) was then calculated according to the contribution of the ion

to the TIC determined from the NIST library. The quantitative data from the ﬁve

.41

replications were averaged and the TIC plotted against time to form curves
depicting the production patterns of the volatiles during the 70 days of the
experiments runs. It is worth notice that TIC does not reﬂect quantity, but it can

be used for identifying trends over time.

RESULTS

In a typical GC run, ester retention times varied approximately between 55
and 200 seconds (Figure 1). Chromatographic separation was not achieved for
many of the volatiles, however determination of the ID unique ions by MS
enabled quantiﬁcation of the responses for many of the target volatiles (Tables 2
and 3).

The initiation of ester biosynthesis followed the climacteric rise in fruit
respiration and ethylene content. Total ester production increased rapidly as
ethylene biosynthesis increased (Figure 2). Immediately after the peak in
ethylene, GCIMS response of total volatiles reached its maximum and then
declined. The peak in the TIC for the individual esters classed by alcohol
occurred on the analysis date following the maximum in ethylene production
(Figures 3a, 4a, 5a, 6a, 7a, 8a, 9a, and 103). However, some esters were
identiﬁed at very low levels prior to the onset of the climacteric (e.g. hexyl, butyl
and 2methylbutyl acetates; butyl and hexyl butanoates; and butyl and hexyl
hexanoates). Typical TICs registered before climacteric were 7x105 for acetates,
9x1 0‘ for butanoates and hexanoates. Ester production increased many folds
during ripening as evidenced by an increase in the TIC to 1x10“, 2x107 and 3x107

respectively.

42

to

esti

for

an

As ripening progressed, the straight chain length of the alcohol-derived
portion of the predominant ester declined. Prior to the onset of the ethylene
climacteric, hexanol esters predominated, although their concentration was very
low. Then, throughout the early portion of the climacteric, butyl esters tended to
predominate. The proportion of propyl esters increased during the late
climacteric and early senescence phase. In late senescence, ethyl ester
proportion increased (Figure 3c). Butanoate, propanoate and 2-methylbutanoate
esters followed similar patterns (Figures 4e, 5e and 7c). The alcohol ester pattern
for acetate esters differed slightly, the trends were much less obvious. Despite
an increasing incorporation of short-chain alcohols, butanol and hexanol, derived
acetate esters maintained their proportions (Figure 6c).

Pentanoate, heptanoate, and octanoate esters were considerably lower
than the ester classes previously discussed (Figures 8,9 and 10). Only propyl
and ethyl pentanoate esters were identiﬁed during the late climacteric and their
levels decreased in early senescence (Figure 8). Butyl heptanoate and butyl
octanoate were identiﬁed early in the climacteric. Propyl and ethyl heptanoate
and octanoate esters predominated later in the climacteric (Figures 9 and 10).
Free hexanol, butanol, propanol, and 2-methylbutanol were detected after the
onset of ethylene climacteric until senescence (Figure 11). Their levels
decreased at the peak in the ethylene climacteric (Figures 3b, 4b, 5b and 7b
respectively). Only free ethanol was detected throughout the experiment yet no
ethyl esters were detected until early senescence (Figures 6b and 11).

Among the compounds present in the standard, 2-methylbutyl acetate had

the highest concentration during ripening and senescence. Its concentration was

43

2800 times higher than its odor threshold (Figure 12a). Only the concentration of
butanol was higher than its odor threshold during senescence (Figure 12 b and

C).

DISCUSSION

The formation of the aroma compounds is Closely correlated with the
metabolic Changes occurring during fruit ripening. Ester GCIMS response
maximized near the peak in ethylene. These ﬁnding agree with those of Song
and Bangerth (1996) and Fan et al. (1998) who determined that normal ester
biosynthesis in apples depends on continuing presence of ethylene. Other
authors also found the peak of apple volatile compounds just after the climacteric
peak (Brown et al., 1966; Dirinck et al., 1989; Mattheis et al., 1991; Tressl and
Drawert, 1973).

The GCIMS response of the esters identiﬁed before the onset of the
autocatalytic increase was low, however, Mattheis et al. (1991), working with
headspace sampling from intact ‘Bisbee Delicious’ fruit, observed also that 2-
methylbutyl acetate preceded the increased ethylene levels associated with the
onset of apple ripening. Butyl acetate and hexyl acetate were present in small
concentrations during growth or at the time of harvest in ‘Golden Delicious’ fruit
and were only produced in higher amounts during ripening (Willaert et al., 1983).
This indicates that the ester biosynthesis system is engaged prior to autocatalytic
ethylene formation and that the alcohol and acid precursors are available before
the ethylene climacteric. Thus, it appears that either the AAT activity is low or the

substrate availability limits ester production in preclimacteric apples. Precursor

44

feeding studies in preclimacteric apple fruit (De Pooter et al., 1983; Knee and
Hatﬁeld, 1981; Song and Bangerth, 1996) demonstrated that the supply of
substrates seems to be the limiting factor, rather than the amount of AAT present
before the onset of ripening. Similar results were found by Jayanty et al. (2002)
in banana fruit. They suggest that the primary limiting factor in ester biosynthesis
before natural production is precursor availability, but, as ester biosynthesis is
engaged, the activity of AAT exerts a major inﬂuence.

The aldehydes (butanal, pentanal, (E)-2-hexenal, and heptanal), and not
the esters, have been found to be the main group of volatile compounds
detectable from intact immature apples (De Pooter et al., 1987; Fellman et al.,
1991, 2000; Flath et al., 1967; Knee and Hatﬁeld, 1981; Mattheis et al., 1991).
However, no aldehydes were detected in ‘RedChief Delicious’ apples in this
study.

The qualitative composition of esters was similar to that found by other
investigators (Brackmann et al., 1993; Mattheis et al., 1991; Rowan et al., 1996;
Song and Bangerth, 1996; Vanoli et al., 1995). The high concentration of 2-
methylbutyl acetate, hexyl acetate and butyl acetate during the early climacteric
are the same as those previously reported for other Delicious cultivars (Berger
and Drawert, 1984; Brackmann et al., 1993; Dimick and Hoskin, 1983; Fellman
et al., 1993, 2000; KakigUChi et al., 1986; Mattheis et al., 1991, 1995). On the
other hand, other compounds such as 3-penten-2-ol associated with the
Characteristic apple-like aroma of ‘Starkspur Golden’ fruit (Vanoli et al., 1995) or
4-methoxyally benzene (Kakiguchi et al., 1986), which has been reported as

contributing to the spice-like aroma in Jonathan, were not detected in this

45

experiment. Although there appears to be some similarity in the major esters
among different apple cultivars (Fellman et al., 2000), the genotype probably
explains the differences found in the quality of the aroma proﬁle of ripe and
overripe apples among different studies.

The decrease in total volatile GCIMS response, as ethylene biosynthesis
declined, could be attributed to a decrease in activity of the enzymes involved in
their biosynthesis, to a lack of substrate availability, or to an enhanced esterase
activity (Figure 1 in Chapter 2).

The pattern in the alcohol portion found in hexanoates, butanoates,
propanoates, and 2-methylbutanoates has not been previously described in
detail for apple fruit. However, some indications of this pattern are evident from
studies by Vanoli et al. (1995) who found a high content of low boiling-point
esters and alcohol later in ripening and by Panasiuk et al. (1980) and VIﬁllaert et
al. (1983) who correlated overripeness with more ethyl esters. The change in the
alcohol pattern may be related with Changing speciﬁcity of the AAT enzyme for
substrate Chain length. But also, this shift in the alcohol portion of the ester with
time could indicate a developmentally dependent change in the availability of
alcohol precursors from predominantly long to predominantly short Chains.

On the other hand, while the chain-length speciﬁcity for apple AAT is
unknown, that for strawberry has a greater preference for acetyl-GOA to form
esters with long-chain alcohols (Perez et al., 1993). This is consistent with ester
proﬁle changes during late climacteric in our study when, despite increasing
incorporation of short-chain alcohols (ethanol and propanol), butanol- and

hexanol-derived acetate esters maintain their proportions. Strawberry AAT has

46

alcohol substrate speciﬁcity in the order hexyl>butyl>amyl>isoamyl using acetyl-
CoA as co-substrate, and acyl-CoA substrate speciﬁcity in the order
acetyl>butyl>>propionyl using butyl alcohol as co-substrate (Perez et al., 1993).

Of all the esters identiﬁed, the ester formed with the branched-Chain
alcohol 2-methylbutanol and acetic acid had the greatest GCIMS response early
in climacteric. Interestingly, this branched-chain alcohol was identiﬁed forming
esters signiﬁcantly only with acetic acid, which may be related with the speciﬁcity
of the AAT enzyme.

The fact that free hexanol and propanol were detected from the onset of
ethylene climacteric until late in senescence suggests that once the ethylene
production starts to increase, the availability of hexanol and propanol is not a
limiting factor for ester formation. The decrease in hexanol and propanol at the
peak in the ethylene climacteric may have been due to higher AAT activity at that
point. Then, as senescence commenced, the activity of AAT declined leaving
unreacted hexanol and propanol. The alcohol ethanol is also not a limiting factor
for the ester biosynthesis given that it was detected prior and throughout ripening
and senescence. The fact that only ethyl esters increased at the end of ripening
and in senescence suggests that AAT preference for ethanol may have
increased or it could be an AAT isoenzyme present during senescence with
higher speciﬁcity for ethanol. No free pentanol or 2-methylpropanol was detected
and the GCIMS response for these esters were very low suggesting that the
availability of the alcohols pentanol and 2methyl-propanol could be a limiting

factor for the biosynthesis of these ester classes.

47

CONCLUSIONS

The ester formation system is present before the onset of the
autocatalytic increase but the formation of the characteristic apple aroma
compounds is correlated with the metabolic changes occurring during ripening
and senescence.

As ripening progressed, there was a change in the alcohol portion of
esters from predominantly long to predominantly short straight chains. This
change may be related with changing speciﬁcity of the AAT enzyme for substrate
chain length, or may indicate a developmentally dependent Change in the
availability of alcohol precursors.

The limiting factor for ester biosynthesis could be substrate availability
before onset of the ethylene climacteric, the level of one precursor relative to the
other during ripening or a shift from substrate limitation to enzyme limitation later
in senescence. Probably there is a different AAT in senescence with higher
speciﬁcity for short Chain alcohols. It could be also possible that acyl-CoA
synthetase activity declines and/or there is an increase of esterase activity in
senescence.

The data obtained in this experiment will be properly interpreted when
more is known about the ester formation system and the family of AAT enzymes.
Enzyme speciﬁcity in apple for acid and alcohol carbon Chain length needs to be

more fully characterized.

48

References

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Synthesis of aroma compounds by apples supplied with alcohols and
methyl esters of fatty acids. J. Sci. Food Agric. 36: 567-574.

Beaudry, R. 2000. Aroma generation by horticultural products: what can we
control? Introduction to the workshop. HortScience 35(6): 1001-1002.

Berger, R. G. and F. Drawert. 1984. Changes in the composition of volatiles by
post-harvest application of alcohols to Red Delicious apples. J.Sci.Food
Agric. 35: 1318-1325.

Brackmann, A., J. Streif and F. Bangerth. 1993. Relationship between a reduced
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Brown, D. 8., JR. Buchanan, and JR. Hicks. 1966. Volatiles from apple fruits as
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De Pooter, H. L., J. P. Montens, G.A. Willaert, P.J. Dirinck, and N.M.Schamp.
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Dimick, P. S. and J. C. Hoskin. 1981. Review of apple ﬂavor - state of the art.
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Dirinck P., H. De Pooter, and N. Schamp. 1989. Aroma development in ripening
fruits, p. 24-34. In: R. Teranishi, R.G. Buttery, F. Shahidi (eds). Flavor
Chemistry: Trends and Developments. ACS Symposium Series (388)
Washington, DC.

Fan X.T., J.P. Mattheis, and D. Buchanan. 1998. Continuous requirement of
ethylene for apple fruit volatile synthesis. J. Agr. Food Chem. 46(5):1959-
1963.

Fellman, J.K., J.P. Mattheis, D.S. Mattinson, and B. Bostick. 1991. Assay of
acetyl COA alcohol transferase in ‘Delicious' apples. HortScience
26(6):773.

Fellman, J. K. and J. P. Mattheis. 1995. Ester biosynthesis in relation to harvest
maturity and controlled-atmosphere storage of apples, p. 149-162. In: R.
L. Rouseff and M.M.Leahy (eds). Fruit Flavors: biogenesis,
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Washington, DC.

49

Fellman, J. K., D. S. Mattinson, B.C. Bostick, J.P. Mattheis, and ME. Patterson.
1993. Ester biosynthesis in ‘Rome' apples subjected to low-oxygen
atmospheres. Postharvest Biol. Tech. 3: 201-214.

Fellman, J. K., T. W. Miller, and 0.8. Mattinson. 2000. Factors that inﬂuence
biosynthesis of volatiles ﬂavor compounds in apple fruit. HortScience
35(6): 1026-1033.

Flath, R. A., D. R. Black, D.G. Guadagni, W.H. McFadden, and TH. Schultz.
1967. Identiﬁcation and organoleptic evaluation of compounds in
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Jayanty, S., J. Song, N.M. Rubinstein, A. Chong, and RM. Beaudry. 2002.
Temporal relationship between ester biosynthesis and ripening events in
bananas. J. Amer. Soc. Hort. Sci. 127(6):998-1005.

Kakiguchi, N., S. Moriguchi, H. Fukuda, N. lchimura, Y. Kato, and Y. Banba.
1986. Composition of volatile compounds of apple fruits in relation to
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971-975.

Knee, M. and S. G. S. Hatﬁeld. 1981. The metabolism of alcohols by apple fruit
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Washington, DC.

50

Perez, A. G., C. Sanz, and J.M. Olias. 1993. Partial puriﬁcation and some
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269.

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esters in bananas (Musa sapientum L.). J. Agric. Food Chem. 48:3493-
3496.

51

 

 

 

 

 

 

 

 

 

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52

Table 2: Volatile compounds (esters, alcohols, and acids) identiﬁed in ‘Redchief
Delicious’ apple fruit during ripening and senescence as a function of GC
retention time (seconds).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

COMPOUND RT (SECONDS)

ethyl acetate 57.4
ethanol 63.3
ethyl propanoate 67.1
propyl acetate 69.9
2-methylpropyl acetate 75.9
propanol 79
ethyl butanoate 79.9
propyl propanoate 81.3
ethyl 2-methylbutanoate 82.7
butyl acetate 86.4
2-methylbutyl acetate 95.2
propyl butanoate 95.5
ethyl pentanoate 97.3
butanol 98
propyl 2-methylbutanoate 98.4
butyl propanoate 98.5
pentyl acetate 1 04.1
2-methylbutyl propanoate 1 06.9
2-methylbutanol 108.3
butyl butanoate 1 12.4
propyl pentanoate 1 12.8
ethyl hexanoate 1 15.2
butyl 2-methylbutanoate 1 15.3
2-methylbutyl butanoate 121.1
Hexyl acetate 122.4
Propyl hexanoate 1 30.4
Pentyl 2-methylbutanoate 131.8
ethyl heptanoate 1 32.5
hexyl propanoate 1 33.6
hexanol 1 34.4

 

53

 

 

Table 2 (cont’d).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

COMPOUND RT (SECONDS)
2-methylpropyl hexanoate 1 35.6
butyl hexanoate 146.6
hexyl butanoate 146.9
propyl heptanoate 147.2
hexyl 2-methylbutanoate 149
ethyl octanoate 149.6
acetic acid 152.6
2-methylbutyl hexanoate 153.8
pentyl hexanoate 162
butyl heptanoate 162.2
propyl octanoate 163.2
propionic acid 166.4
hexyl hexanoate 177.6
butyl octanoate 178
butanoic acid 180.2
2-methylbutanoic acid 186.4
hexanoic acid 212.7

 

 

 

Table 3. Conversion factors for those volatile compounds present in the

 

 

 

 

 

 

 

 

 

 

standard.

COMPOUND CONVERSION FACTOR
ethanol 3.06X10‘5
butyl acetate 2.60X10'8
2-methylbutyl acetate 8.92X1 0'8
butanol 1.16X10'7
2-methylbutanol 5.67X104’
hexyl acetate 8.93X1 0‘9
hexanol 1 .47X10‘
hexyl butanoate 6.06X10'9
hexyl hexanoate 1.65X10f

 

 

 

 

55

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Figure 1. Representative gas chromatograph of the headspace of ‘Redchief
Delicious’ apples at climacteric. Most predominant ester peaks are
identiﬁed by numbers: 1. butyl acetate; 2. 2-methylbutyl acetate; 3. hexyl
acetate; 4. hexyl 2-methylbutanoate.

56

 

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Day

Figure 2. Ontogeny of total esters, ethylene and respiration (CO2 production)
during ripening and senescence 0f ‘Redchief Delicious’ apples. The
volatile proﬁle of apple fruit was tracked from 3 weeks prior to eight weeks
after the onset of the ethylene climacteric (indicated by dashed vertical
line). Each symbol represents the average of 5 replications.

57

Figure 3. Pattern of hexanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total hexanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, 2-methylpr0pyl, butyl, 2-methylbutyl, pentyl, and hexyl esters of
hexanoic acid. (C) Ester proportions (% of total hexanoate esters). Each
symbol represents the average of 5 replications.

58

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59

Figure 4. Pattern of butanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total butanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, butyl, 2-methylbutyl, and hexyl esters of butanoic acid. (C) Ester
proportions (% of total butanoate esters). Each symbol represents the
average of 5 replications.

60

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61

Figure 5. Pattern of propanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total propanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, butyl, 2-methylbutyl, and hexyl esters of propanoic acid. (C) Ester

proportions (% of total propanoate esters). Each symbol represents the
average of 5 replications.

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63

Figure 6. Pattern of acetate esters during ripening and senescence of ‘Redchief
Delicious' apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total acetate esters (B)
GCIMS response (TIC) of ethyl, propyl, 2-methylpropyl, butyl, 2-
methylbutyl, pentyl, and hexyl esters of acetic acid. (C) Ester proportions
(% of total acetate esters). Each symbol represents the average of 5
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65

Figure 7. Pattern of 2-methylbutanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total butanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, butyl, pentyl, and hexyl esters of 2-methylbutanoic acid. (C) Ester
proportions (% of total 2-methylbutanoate esters). Each symbol
represents the average of 5 replications.

66

 

 

 

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67

 

Figure 8. Pattern of pentanoate esters during ripening and senescence of
‘Redchief Delicious' apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total pentanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl, and
propyl esters of pentanoic acid. (C) Ester proportions (% of total

pentanoate esters). Each symbol represents the average of 5 replications.

68

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Figure 9. Pattern of heptanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total heptanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, and butyl esters of heptanoic acid. (C) Ester proportions (% of total
heptanoate esters). Each symbol represents the average of 5 replications.

70

 

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Figure 9.

71

Figure 10. Pattern of octanoate esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total octanoate
esters and ontogeny of ethylene. (B) GCIMS response (TIC) of ethyl,
propyl, and butyl esters of octanoic acid. (C) Ester proportions (% of total
octanoate esters). Each symbol represents the average of 5 replications.

72

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73

 

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Figure 11. Pattern of alcohols identiﬁed during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric which
corresponds to the harvest date (indicated by an arrow). Each symbol
represents the average of 5 replications.

74

 

 

 

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Figure 12. Esters and alcohols content during ripening and senescence of
‘Redchief Delicious’ apple fruit. (A) Concentration of 2-methylbutyl
acetate, butyl acetate, hexyl acetate, hexyl hexanoate and hexyl
butanoate esters. (B) Concentration of ethanol, butanol, 2-methylbutanol
and hexanol alcohols. (C) Concentration of butanol, 2-methylbutanol and
hexanol alcohols. The odor threshold for 2-methylbutyl acetate is 0.005
uL/L, for butyl acetate 0.066 uUL, for hexyl acetate 0.002 uL/L, for
ethanol 100 pUL, and for butanol and hexanol is 0.5 uL/L (indicated by
dashed horizontal line) (Flath et al., 1967). Each symbol represents the
average of 5 replications.

75

CHAPTER IV:

PATTERNS IN THE ACID PORTION OF ESTERS PRODUCED DURING RIPENING AND
SENESCENCE OF ‘REDCHIEF DELICIOUS, APPLE FRUIT.

76

INTRODUCTION

Flavor is an important parameter of fruit quality that inﬂuence consumer
acceptability. Aroma compounds contribute signiﬁcantly to the ﬂavor of all fresh
fruits. However in recent years, the retail industry and consumers recognize
apple ﬂavor as needing improvement (Beaudry, 2000). The aroma of apple fruit
depends on the concentration of a complex mixture of low molecular weight
esters, alcohols, aldehydes, and hydrocarbons. More than 300 volatile
compounds have been identiﬁed in apple (Dimick and Hoskin, 1981) being the
esters the major constituents.

Aroma biosynthesis is affected by many factors that impact the ester
biosynthesis: cultivar, growing conditions, fruit maturity, and also storage
conditions (Dirinck, et al., 1989; Fellman, 2000; Mattheis et al., 1991). Fruit
maturity is probably one of the most significant factors (Song and Bangerth,
1996). It is known that aroma biosynthesis is correlated with ethylene synthesis
and action (Brown et al., 1966; Dirinck et al., 1989; Mattheis et al., 1991; Song
and Bangerth, 1996; Tressl and Drawert, 1973). However, it is not clear whether
the onset of biosynthesis of volatile compounds is concurrent with, or precedes
the climacteric rise in fruit respiration (Fellman et al., 2000).

Esters are formed from fatty acids and alcohols (Figure 1 in Chapter 2).
The enzyme alcohol acyl-CoA transferase (AAT, EC2.3.1.84) catalyzes the union
of an alcohol and the acyl-CoA derivative of a fatty acid. The carbon chain length
of the alcohol portion of the ester varies between 2 and 6 carbons and the acid
portion has typically a chain length from 2 to 8 carbons. The carbon chains of the

alcohols or fatty acids can be straight or branched. The branched-chain

77

compounds are believed to be derived from amino acids (Nursten, 1970; Perez
et al., 1992; Tressl and Drawert, 1973; Wyllie, et al., 1995). The straight short-
chain fatty acids (SSCFA), between 2 and 8 carbon length, are believed to be
derived from fatty acid metabolism, either degradation (Bartley et al., 1985;
Brackmann et al., 1993; Fellman et al., 2000; Nursten, 1970; Rowan et al., 1996,
1999) or synthesis (Tan and Bangerth, 2001).

Ester biosynthesis could be inﬂuenced by substrate availability (Knee and
Hatﬁeld, 1981; Wyllie and Fellman, 2000), AAT speciﬁcity (Olias et al., 1995;
Perez et al., 1993), AATexpression (Aharoni et al., 2000a; Jayanty et al., 2002),
and/or AAT activity levels (Perez et al., 1996). The importance of these factors
appears to change as ripening progresses. It is not known how many different
AAT isozymes could be present in apples. Two AAT genes have been recently
cloned from strawberry, (Aharoni et al., 2000a, 2000b) and one gene has been
identiﬁed in banana and apple (Aharoni et al., 2000b). No study on apple AAT
activity during fruit development has been published; only preliminary studies on
the effect of different storage conditions on AAT activity has been carried out
(Fellman et al., 1993; Fellman and Mattheis, 1995; Ke et al., 1994).

Although considerable progress has been made in isolating and identifying a
large number of volatile compounds from plant aromas, less work has been done
on elucidating the aroma formation mechanism.

The aim of this research was to characterize the patterns in ester
biosynthesis during ripening and senescence of ‘Redchief Delicious’ apple to
better understand the biochemical origin and fate of these organoleptically

signiﬁcant compounds. Apple fruit were tracked throughout ripening and selected

78

fruit for analysis based on internal ethylene levels. At each stage evaluated,
respiration and ester production was measured for ﬁve representative fruit.
Developmentally dependent patterns in esters were evaluated. An ester matrix
was established based on precursor acids and alcohols (T able 1 in Chapter 3).
One axis of the matrix included alcohols (ethanol, propanol, 2-methylpropanol,
butanol, 2-methylbutanol, pentanol, and hexanol) and the other axis acids
(acetate, propanoate, butanoate, 2-methylbutanoate, pentanoate, hexanoate,
heptanoate, and octanoate). Few alcohol/acid combinations were not detectable.
In this paper, we focused on patterns evident in the acid portion of esters

classed by the alcohol moiety during ripening and senescence.

MATERIALS AND METHODS

‘Redchief Delicious’ apples [Malus sylvestn’s (L) Mill. var. domestica
(Borkh.) Mansf.] were harvested every three to four days at the Michigan State
University Horticultural Teaching and Research Center, East Lansing, MI,
beginning three weeks prior to the onset of the climacteric and continuing until
fruit were considered to have initiated ripening based on internal ethylene
content (IEC). The beginning of the climacteric rise was considered to occur
when the internal ethylene content was about 0.2 uL/L. The harvest date
occurred on October 3", which was day 25 of the experiment. Distinct patterns in
the ester production were evident.

After the initiation of ripening, the remaining fruit were harvested and held
at room temperature for analysis continuing fruit selection for 45 days. Thus, 18

different stages of development of ‘Redchief Delicious' apple fruit ranging from

79

unripe through senescent over a period of 70 days were measured. The average
IEC of twenty representative fruits at each stage was determined and those ﬁve
fruit nearest the average were chosen for ester evaluation.

The IEC was determined by withdrawing a 1-mL gas sample from the
interior of apples and subjecting the gas sample to gas chromatographic (GC)
analysis. The gas chromatograph (Carle Series 400 AGC; Hach Co., Loveland,
Colo.) was ﬁtted with a 6-m-Iong, 2-mm—i.d. stainless-steel column packed with
activated alumina and detection was via a ﬂame ionization detector. The
ethylene detection limit was approximately 0.005 uL.L“. Ethylene concentrations
were calculated relative to a certiﬁed standard (Matheson Gas Products,
Chicago, III.) with an ethylene concentration of 0.979 pL.L“.

Volatile analysis procedure was done as described by Song et al.
(1997,1998). Ester emissions were sampled by sealing one fruit in each one of
ﬁve 1-liter Teﬂon TM chamber. In order to reach a steady-state concentration of
apple fruit volatiles in the headspace over the apples, the fruit were maintained
in the chambers for approximately three hours at 22°C and the chambers were
ventilated with pure air at a rate of approximately 30 mUmin. One chamber with
no fruit was used as a blank.

A 1-cm long solid-phase microextraction (SPME) ﬁber coated with a film
thickness of 65 pm of polydimethylsiloxane/divinylbenzene (Supelco Co.,
Bellefonte, PA) was used to adsorb the volatile sample. The SPME ﬁber was
preconditioned by baking overnight at 260°C.

The ﬁber was manually inserted through a Teﬂon-lined half-hole septum

into a glass ‘tee’ located at the outlet of the chambers. Once in the glass ‘tee’

80

outlet, the ﬁber was extended to absorb volatiles for ﬁve minutes. The ﬁber was
then retracted prior to removal from the sample container.

Ester analysis was by GCItime-of-ﬂight mass spectrometry (MS). The
SPME ﬁber was inserted in the glass-lined, splitless injection inlet of the GC
(230'C) and desorbed for 5 minutes. The volatiles were cryofocussed oncolumn
using a liquid nitrogen cryo trap.

The desorbed ﬂavor compounds were separated by a Hewlett-Packard
6890 GC with a capillary column (Supelcowax, 15 m X 0.1 mm i.d., 0.25 pm
coating ﬁlm) (Supelco Co. Bellefonte, PA). The temperature of the GC was
programmed from 40 to 240 °C at 50 'C/min. A constant mass ﬂow rate (0.5
mL/min) of the carrier gas (He) in the column throughout the run was maintained.

The identiﬁcation and quantiﬁcation of the volatiles were by comparison
with the National Institute of Standards and Technology database and
authenticated standards. Quantiﬁcation for selected compounds was
accomplished using gas standards. Gas standards were created from a mixture
of equal volumes of the neat oils of 13 compounds. A sample of 0.5 uL was
taken by using a Hamilton 1.0 uL syringe, which was discharged onto a ﬁlter
paper disk. The ﬁlter paper was immediately dropped into a 4.4-L glass
volumetric ﬂask ﬁtted with a ground-glass stopper containing a gas-tight Mininert
valve (Alltech Assoc., Inc., Deerﬁeld, IL). A new standard was made every
month. Volatile aroma compounds were purchased from Sigma Co. and F luka
Chemical Corp. The compounds included in the standard were: 1-butanol, 1-
hexanol, cis-3—hexen-1-ol, ethyl alcohol, acetaldehyde, 1-methyl-1-butanol, n-

butyl acetate, hexyl acetate, hexyl butyrate, hexyl hexanoate, 3-methylbutyl

81

acetate, 2-methylbutyl acetate, and famesene mixture. For all compounds
identiﬁed, not all standards were available.

For each sample, all target compounds were identiﬁed. The peak area
was determined under the unique ion ID for each speciﬁc compound and the
total ion count (TIC) was then calculated according to the contribution of the ion
to the TIC determined from the NIST library. The quantitative data from the ﬁve
replications were averaged and the TIC plotted against time to form curves
depicting the production patterns of the volatiles during the 70 days of the
experiments runs. It is worth notice that TIC does not reﬂect quantity, but it can

be used for identifying trends over time.

RESULTS

In a typical GC run, ester retention times varied approximately between 55
and 200 seconds (Figure 1 in Chapter 3). Chromatographic separation was not
achieved for many of the volatiles, however determination of the ID unique ions
by MS enabled quantiﬁcation of the responses for many of the target volatiles
(Table 2 in Chapter 3). Among the substances found, 38 esters, 5 alcohols and 5
acids were identiﬁed and quantiﬁed by GCIMS (Table 2 in Chapter 3). According
to the GCIMS response, acetate esters were the most abundant compounds.
Butyl, hexyl and 2-methylbutyl acetate esters predominated. The branched ester
hexyl 2-methylbutanoate was also among the most abundant esters.

Total ester volatiles reached maximum levels at a time, which nearly

coincides with the peak in ethylene content and respiratory climacteric (Figure 2

82

in Chapter 3). Then, as ethylene biosynthesis declined so too did total volatile
ester biosynthesis.

The peak in the TIC for the individual esters classed by the acid moiety
occurred on the date following the maximum in ethylene production (Figures 1a,
2a, 3a, 4a, 5a, 6a and 7a).

Some esters were identiﬁed in small concentration during growth or at the
time of harvest and were only produced in higher amounts during ripening (e.g.
hexyl, butyl and 2methylbutyl acetates; butyl and hexyl butanoates; and butyl
and hexyl hexanoates).

Proportionally, the acetate esters of hexanol, butanol, 2-methylbutanol,
propanol, 2-methylpropanol and pentanol predominated prior to the onset of the
ethylene climacteric (Figures 1c, 2c, 3c, SC, SC and 7c). As ripening progressed,
acetate esters decreased while all the other acids increased in their proportions.
During senescence, acetate esters predominated again having the highest
proportion. In the case of ethyl esters, not only acetate esters of ethanol, but also
all acids increased their proportions in the late stages of senescence (Figure 4c).

The TIC for hexyl and butyl esters exhibited a broad peak earlier in
climacteric while propyl and ethyl esters all peaked after the ethylene climacteric
peak (Figures 1b,2b, 3b and 4b respectively).

The TIC for esters formed with pentanoic acid and longer chain acids like
heptanoic and octanoic acids, was considerably low (Figures 1b,2b,3b, 4b and
5b).

Free hexanoic and propanoic acids were detected after the respiration

and ethylene climacteric peaks, when the synthesis of total hexanoic and

83

propanoic esters started to decline (Figure 8). Free butanoic and 2-
methylbutanoic acids were detected from the onset of ethylene climacteric and
they declined in senescence (Figure 8). Free acetic acid was found throughout
the experiment. All free acids declined at the ethylene climacteric peak. No
pentanoic, heptanoic or octanoic acids were detected.

Some alcohols increased late in ripening (ethanol, propanol, hexanol), as
did acids (hexanoic, propanoic, acetic acid), but the formation of the associated
esters did not increase with the exception of propyl and ethyl acetates (Figures 9

and 10).

DISCUSSION

The qualitative composition of esters was similar to that found by other
investigators (Brackmann et al., 1993; Fellman et al., 2000; Mattheis et al., 1991;
Rowan et al., 1996; Song and Bangerth, 1996; Vanoli et al., 1995). Acetate
esters were the most abundant compounds probably because acetyl CoA is the
most abundant acyl CoA present in fruit tissue as it is explained by Nursten
(1970).

The increase in ester emanation following the onset of the ethylene
climacteric is consistent with the ﬁndings of Song and Bangerth (1996) and Fan
et al. (1998) who determined that normal ester biosynthesis in apples depends
on continuing presence of ethylene. The increase in respiration may be related
to an increase in substrate availability. Song and Bangerth (1996) suggested that
more a general and not a speciﬁc increase in metabolic activity is a prerequisite

for the stimulation of aroma production. Bangerth et al. (1998) argued that is

84

rather unlikely that ethylene directly affects the production of so many individual
volatile substances. They suggest that ethylene determines an increase of fruit
respiration which provides the necessary energy (ATP, NADPH, etc.) for the
synthesis of aroma volatile precursors.

The fact that free butanol and ethanol were detected prior the onset of
ethylene climacteric but only few esters were identiﬁed quite low, suggests that
AAT activity may be limiting at this point of time. Other possibilities are that acid
availability and/or the conversion of acids to acyl-CoA are limiting. The
possibility that acid availability is limiting factor for ester biosynthesis before the
onset of ethylene climacteric has been observed previously (Berger and Drawert,
1984; Fomey et al., 2000; Knee and Hatﬁeld, 1981; Williams and Knee, 1977).
Treatments of Golden Delicious apples with aldehydes and carboxylic acids
suggest that there is a certain selectivity of the apple AAT in the use of the
carboxylic acid precursors (De Pooter et al., 1983). They supported the
hypothesis that the composition of apple aroma is determined by not only the
availability of acids but also by their identity. It could be also a different AAT
isoenzyme present before the onset of ethylene climacteric with a lower
speciﬁcity for ethanol since no ethyl esters were detected until late in climacteric.

A pattern in the alcohol portion was found in all but the acetate esters as
ripening progressed (discussed in ﬁrst paper). There was a change in the alcohol
moieties in the esters predominantly long to predominantly short chains as fruit
ripened. A similar pattern was not observed in the chain length of the fatty acid

portion. This fact suggests separate pathways for the substrates for acids and

85

alcohols or at least no free interconversion by acyl-COA reductase or other
enzyme system.

The decline in free acetic and butyric acids at the ethylene peak suggests
that AAT activity achieved its maxima at the ethylene climacteric peak coinciding
with the peak in total esters production. At later stages both acids and alcohols
are in abundance, yet total ester formation declines. It is possible that the activity
of AAT declines during senescence leaving unreacted free acids and alcohols.
An increase in the activity of the esterase enzyme during senescence is also a
possibility.

The fact that some free alcohols and acids increased late in ripening but
the formation of the associated esters did not increase suggests that later in
senescence there is a shift from substrate limitation to enzyme limitation.
Probably there is a different AAT in senescence with higher speciﬁcity for short
chain alcohols and/or acyl-COA synthetase activity declines in senescence. It
could be also an increase of esterase activity in senescence. Late in ripening,
long- and medium-chain alcohols formed carboxylic esters with acetic acid, and
long- and medium-chain fatty acids formed esters with ethanol. This observation
suggests that other AAT isoenzyme could be present during senescence with
different inherited properties that determines a limit in the number of carbons of

the ester molecule.

86

CONCLUSIONS

Ester formation requires acyl-COA, which is associated with the
fundamental metabolism of the cell. This could explain the association of the
climacteric in fruits with maximum levels of esters. However, ester precursors as
well as AAT activity are present before the increase in metabolic activity.

Although further work should be done, this study allow us to suggest that
different factors are involved in determining volatile ester composition in
‘Redchief Delicious’ apple fruit: acid and alcohol availability, AAT activity and
inherent characteristics; acyl-COA synthetase activity as well as esterase activity.

Ester formation depends on the availability of alcohols and CoA-
derivatives. Thus, both alcohols and acids compete in ester biosynthesis.

The data of this experiment also suggests that there are separate
pathways for the substrates for acids and alcohols or at least no free
interconversion by acyl-CoA reductase. Whether different AAT isoenzymes

predominate at different times during ripening and senescence is not known.

87

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91

Figure 1. Pattern of hexanol esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total hexanol esters
and ontogeny of ethylene. (B) GC/MS response (TIC) of acetic, propanoic,
butanoic, 2-methylbutanoic, and hexanoic esters of hexanol. (C) Ester
proportions (% of total hexanol esters). Each symbol represents the
average of 5 replications.

92

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93

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Figure 2. Pattern of butanol esters during ripening and senescence of ‘Redchief
Delicious' apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total butanol esters
and ontogeny of ethylene. (B) GCIMS response (TIC) of acetic, propanoic,
butanoic, 2-methylbutanoic, hexanoic, heptanoic, and octanoic esters of
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Figure 3. Pattern of propanol esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total propanol esters
and ontogeny of ethylene. (B) GCIMS response (TIC) of acetic, propanoic,
butanoic, 2-methylbutanoic, pentanoic, hexanoic, heptanoic and octanoic
esters of hexanol. (C) Ester proportions (% of total propanol esters). Each
symbol represents the average of 5 replications.

96

 

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97

Ethylene (/.1L-L")

Figure 4. Pattern of ethyl esters during ripening and senescence of ‘Redchief
Delicious’ apple. The volatile proﬁle was tracked from 3 weeks prior to
eight weeks after the onset of the ethylene climacteric (indicated by
dashed vertical line). (A) GCIMS response (TIC) of total ethyl esters and
ontogeny of ethylene. (B) GCIMS response (TIC) of acetic, propanoic,
butanoic, 2-methylbutanoic, pentanoic, hexanoic, heptanoic and octanoic
esters of ethanol. (C) Ester proportions (% of total ethyl esters). Each
symbol represents the average of 5 replications.

98

 

 

 

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Figure 5. Pattern of 2-methylbutanol esters during ripening and senescence 0f
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total 2-
methylbutanol esters and ontogeny of ethylene. (B) GCIMS response
(TIC) of acetic, propanoic, butanoic, and hexanoic esters of 2-
methylbutanol. (C) Ester proportions (% of total 2-methylbutanol esters).
Each symbol represents the average of 5 replications.

100

 

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101

Figure 6. Pattern of 2-methylpropanol esters during ripening and senescence of
‘Redchief Delicious’ apple. The volatile proﬁle was tracked from 3 weeks
prior to eight weeks after the onset of the ethylene climacteric (indicated
by dashed vertical line). (A) GCIMS response (TIC) of total 2-
methylpropanol esters and ontogeny of ethylene. (B) GCIMS response
(TIC) of acetic, and hexanoic esters of 2-methylpropanol. (C) Ester
proportions (% of total 2-methylpropanol esters). Each symbol represents
the average of 5 replications.

102

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103

Figure 7. Pattern of pentanol esters during ripening and senescence of ‘Redchief
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dashed vertical line). (A) GCIMS response (TIC) of total pentanol esters
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106

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Figure 9. Pattern of hexanol, hexanoic acid and the associated ester (hexyl
hexanoate) during ripening and senescence for ‘Redchief Delicious’ apple
fruit. Each symbol represents the average of 5 replications.

107

 

 

 

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Figure 10.Pattern of ethanol, acetic acid and the associated ester (ethyl acetate)
during ripening and senescence for ‘Redchief Delicious’ apple fruit. Each
symbol represents the average of 5 replications.

108

APPENDIX

109

Table 1. Original data of Figure 2 (Chapter 3). Ethylene content and CO2
production. Each value is the average of 5 replications.

 

Date Ethylene (uLIL) STD COZ (nmollkgg) STD

 

0 0.0384 0.00368 65.5 36.5
6 0.108 0.0518 70.3 4.92
9 0.0530 0.00580 69.1 6.77
12 0.0455 0.0155 68.6 5.82
16 0.0843 0.0156 97.4 8.57
19 0.122 0.143 84.5 5.91
21 0.0404 0.0205 71.6 6.81
25 0.499 0.337 106.8 10.9
29 52.3 13.6 134.8 19.3
33 120.6 16.8 128.2 9.31
37 150.1 4.27 154.2 11.8
41 219.2 17.2 147.7 12.8
44 262.0 17.6 154.2 12.0
47 302.0 19.7 151.7 5.33
49 232.8 8.98 121.3 15.6
55 218.8 9.78 133.6 6.85
63 130.3 14.5 102.9 33.4
71 46.6 11.4 86.7 6.72
86 38.4 4.32 98.4 14.7

 

110

 

 

 

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