Postharvest improvement of Cavendish banana quality and ...

t6.tz. oz

Postharvest improvelnentof Gavendislr bananagualit¡r and shelf life

Annunziata (Nancy) Teresa Bagnato

B.Ag.Sc. (Ilorticulhral Science, Hons. The University of Adelaide)

A thesis submitted in fulfilment of the

requirements forthe degree of

Iloctoro,f PHlosophy

The University of Adelaide

Departrnent of Horticulture, Viticulture and Oenology

Facuþ ofSciences

Waite Campus

November 2002

Table of Contents

TABLE OF CONTENTS

Ansrnlcr

Drcr,¡,mrroN........

IV

III

IX

XI

Lrsr on Frcrnns...........

LrsrorTABr,ns

Ac¡crowr.nDcMENTS...........

ArnnnvnrroNs...... XItr

CHAPTER 1........ 1

1. Gn¡wn¡,r,I¡rrRoDUCTIoN

CHAPTER 2.

1

...................4

2. LrrrnrrunnRnvrew....

2.1 Banana classification

2.2 Climactericfruit.

2.3 The ripeningprocess of bananas.....

2.4 Temperature in relation to ripening

2.4.1 Low temperatures

2.4.1.1 Chlorophyll fluorescence

2.4.1.2 Polyphenoloúdase activity in chilled fruit

4

5

4

5

10

t4

l6

.. 11

I

Table of Contents

2.4.1.3 Peroxidase activrty.........

2.4.2 High temperature...........

2.5 Ethylene in relation to ripening-...'-.

2.6 Humidity in relationto ripening......

2.7 Post ripening storage

3.I

3.2

3.3

3.4

19

19

2.7 .1 Controlled atrnosphere and modified atmosphere storage ........23

2.7 .l.l Nifogen atnosphere storage.'....... """""""24

2.7 .1.2 Ethanol aÍnosphere storage

2.7.2 Ethylene antagonist 26

2.8 Summary... 30

2.9 Research aims..... 31

22

25

Plant material.....

P re parat ion of fruit pri or t o expe rim ent at i on ... -.

Monitoring ethylene levels .........

Monitoring carbon dioxide ønd oxygen in storage atmospheres

3.5 External quality assessment....

3.5.1 Green life

)z

32

JJ

a1JJ

34

343.5.2 Shelf life

Table of Contents

3.5.3 Discolouration index

3.5.4 Weight loss ............

3.6 Intemal quality assessments

3 .6 .1 Pulp firmness . .. . . . .. . .. .. . ..

3.6.2 Soluble solid content

3.6.3 Aroma analysis

3 .6.3 .l Chemical nose statistical analysis ... ... ...... ..

CITAPTER 4.........

4. Pnn-NTPUXNGDETECTION OF CIilI ,NGINJURY

4.1 Introduction.

4.2 Materialsandmethods........

4.2.1 Chlorophyllfluorescence

4.2.2 Pollphenoloúdase activity

4,3 Results.......

4.3.1 Chlorophyllfluorescence

4.3.2 Polyphenoloxidase...........

4.4 Discussion.

4.5 Conclusion

..36

38

38

38

41

4t

42

44

44

44

46

48

50

50

53

54

57

Table of Contents

5. OPTNTNSTXGRIPENIINGTEIæNN¡.TURESA¡{DETI{YIJNE

CoNCnNTUTTONS THROUGHOUT TTru YEAR. .......58

5.1 Introduction 58

5.2 Materials and methods...

s.2.1

s.2.2

s.2.3

s.2.4

s.2.5

Plant material and procedure

Effect of ripening temperature and harvest months...".

Effect of ethylene concentrations and harvest months.

Quality assessments

Statistical assessment

59

59

60

60

61

61

62

62

64

75

83

88

5.3 Results

5.3.1 Climatic data for Tully, North Queensland

5 .3 .2 Effect of ripening temperature and harvest months.......'.'......

5.3.3 Effect of ethylene concenfiations and harvest months

5.4 Discussion.

5.5 Conclusion

6. Posr-nrpnxncc: NrrnocrN ArMosPrrERE ExPosuRE ...................... 89

6.1 Introduction ...""'89

Table of Contents

6.2 Materials andmethods """"'90

6.2.I Plantmaterial andpreparation """""""90

6.2.2 Nitrogen application for various periods of time at different

ripening stages

6.2.3 Nitrogen atrnosphere storage in the presence of low or high

relative humidiq, 9

6.2.4 Assessments...

6.3 Results 93

6.3.1 Nihogen application for various periods of time at difÊerent

npemng stages

6.3.2 Nitrogen atnosphere exposure in the presence of low or higþ

93

relative humidtty 99

6.4 Discussion........ ...102

6.5 Conclusion 106

7. Posr-nrpnNnlc: ETHANoLAruosprmREExPosuRE ..'108

9 1

1

7.1 Introduction

7.2 Materials and methods

7 .2.1 Plant material and preparation.'..'

7.2.2 Determination of ethanol volume

108

109

110

Table of Contents

7 .2.3 Effect of ethanol vapour and holding times after repressurising

on the quahfy andripening of bananas

7.2.4 Application of ethanol atmosphere

7.2.5 Volatileanalysis

7 .2.6 Banana and statistical assessments.....

7.3 Results.......

8.1 Introduclion...


8.2.1

8.2.2

8.2.3

8.2.4

Plant material and preparation

I -Methylcyclopropene exposure after ethylene initiation

l-MCP detection....

Banana assessment

8.2.5 Statistical analysis ........

8.3 Results

8.4 Discussion

110

112

113

..tr4

115

7.4 Discussion 119

7.5 Conclusion .. t21

8. Posr-nrpnxnc: l-Mnrrrvr-cYCLoPRoPENE ExPosuRE .........----.... 122

122

123

723

124

124

t2s

125

126

t3s

1378.5 Conclusion

Table of Contents

9. Gnxnnlr.DrscussroN..

9.1 Background into industry requiremenls.....'....".... ...138

9.2 Pre-ripening detection of chilling tniury.... .. '....-... 139

9.3 Optimisingripeningtemperatures and ethylene concentrations

throughout the year..... ..........'.-........ 140

9.4 Post-climacteric extension of shelf \fe.............. --....' 142

138

9.4.1

9.4.2

9.4.3

Nitrogen atrnospheres........................

Ethanol afnospheres ...... .. ... ........ .. .. ..

1 -Methylcyclopropene atrnospheres.

Commercial b enefits from this research...

Future research

...142

143

144

9.5

9.6

(a) PUBLICÄTISNS FR6M TIIIS TIIESIS (nnrnrNrs L6CATED AT BACK).150

,Iournal articles....... .-...150

Conference abst racts..... ...1s/1

Miscellaneous

lsz

Abstract

Abstract

Banana quality and shelf life are variable and often inzufficient for marketing.

This study examined the effect of ripening temperatures and ethylene

concenüations t}roughout the year on quality and shelf life. To frlrther extend

banana shelf life, when fruit are eating ripe, post-climacteric exposure to

nitrogen, ethanol or I -methylcyclopropene atrnospheres was studied.

Additionally, detection of chilling exposure in unripe fruit was examined before

visual symptoms developed in order to guide ripening and marketing decisions.

Techniques examined include chlorophyll fluorescence and measuring the

polyphenoloxidase @PO) activity in bananas.

All Cavendi sh cv. II/illiams bananas utilised in the study were grown on a

single property in Tully, North Queensland, harvested at7 5%o maturity when

they were hard and green and transported above 13"C to Adelaide. Pre-

climacteric bananas were exposed to temperatures of 5oC for 4 or 5 days or

stored at room temperature (22"C),but neither chlorophyll fluorescence nor

PPO activity varied in response to chilling injury.

Ripening bananas at 14, 16, I 8 or 20"C with 300 pL Lt of ethylene gas until

they reached colour stage 4 (more yellow than green) had an impact on fruit

quality and shelf life at22"C that varied with the harvesting months of January,

March, May, July, September or November. Shelf life of bananas ripened at 14

and l6"C were extended by up to 50Yo and32%o,respectiveþ, but peel

discolouration also increased, especially on bananas that grew and developed

duringwinter or experienced cyclones. Those ripened at 18 or 20'C throughout

the year averaged a6 day shelf life with consistently lowpeel discolouration.

1

Abstract

Other fruit quality parameters (lirmness, sugar content and aroma) were

generally unaffected.

All ethylene concentrations studied resulted in quality fruit. Ripening fruit at

16"C using 50, 300 or 1000 pL L1 of ethylene did not affect fruit quality or

shelf life, but the use of 50 pLLl of ethylene would minimise commercial

ethylene costs.

Post-climacteric bananas \ùere exposed to a nihogen atmosphere for 6, 12 or 24

hours at ripening stage 3 or 4. Nihogen atmospheres did not extend banana

shelf life, but did cause brown patches on the peel, which increased with

exposure time. The aroma of bananas was also affected, with over-ripe

('alcoholic') profiles detected in fully yellow fruit exposed to nitrogen

atmospheres, compared to the ripe ('fruity') profiles detected in control fruit.

Vacuum infiltration of ethanol afinospheres after ethylene-initiation at 18"C did

not extend banæra shelf life and did not affect the quality of ripening bananas.

However, vacuum infiltation was unsuccessful in forcing ethanol into the pulp,

with increases in ethanol only found in the peel of fruit.

Doubling of banana shelf life occurred with exposure to an atmosphere of 300

nL Ll l-methylcyclopropene (I-MCP) after ethylene-induced ripening at20"C

without negativeþ affecting banana quality, compared to control fruit. Fruit

treated with 30,000 nL Ul I-MCP were unacceptable for human consurnption

as they developed crown rot before full ripening and a lower level of 3 nL Lt l-

MCP had no effect on shelf life.

1r

Decla'ration

Declaration

I, ANNUNZIATA (NANCÐ TERESA BAGNATO, certfi that this thesis

does not incorporate without acknowledgement anymaterial submitted for the

award of any degree or diploma in any university or other tertiary institution,

and that to the best of my knowledge, contains no material previously published

or written by any other person, except where due reference is made in the text.

I give consent to this copy of my thesis, when deposited in the University

Library, being available for loan and photocopying.

2oF Nlov 2"Q

Annurziata Q{anÐ Teresa Bagnato Date

lll

List ofFieures

List of Figures

Number Pøge

Figure 2.lzBananaresponses to temperatures below l2'Cthatlead to the

appearance of chilling injury on fruit (after Wills et a1., 1998)... '. '...... ' . '13

Figure 2.2zThereaction mechanism for oxidation of dopamine bybanana

polyphenoloxidase (Palmeq 1963). . ...18

Figure 2.3: Simplified ethylene biosynthetic pathway in ripening fruit (after

Wang, 1989). -....21

Figure 2.4: Tlte structure of l-methylcyclopropene... . . . . - - -. ....28

Figure 2.5: Model of ethylene and I-MCP probable action on the ethylene

receptor. Elecfion donormetal (M) with ligands of tmknown sfucture (L)

binds to ethylene (E) or I-MCP. Ethylene completes its electron transfer

and is released leavingthe resulting complex active while I-MCP binds so

tightly to the complex it remains attached resulting in an inactive complex

(Sisler and Serek, 1997). . ' .. - ...28

Figure 3.1: The 8 ripening colour stages (CSIRO, 1972). . ... .. . .35

Figure 3.2: The twist tester. . -. -.. . '..40

Figure 4.1: Peel appearafìce of Cavendish bananas cv. Williams after a holding

periodof 5 daysat(Ð5'Cand @)22"C. ............51

Figure 5.1: The daily minimum air temperatures ("C) from South Johnstone

Research Station @ureau of Meteorolory, 2000-01) for the month prior to

harvest. Minimum daily temperatgres were not recorded on days missing

appropriate symbols. .. ... '.. .. '.63

Figure 5.2: Green life (ripening stage l4) of Cavendish bananas cv. Williams

harvested bimonthly and ripened at 14,16,18 or 20'C with 300 pL Lt

lV

List ofFþures

ethylene. Each column represents the mean of 3 replicates consisting of 6

bananas each; the bar denotes least significant differences between

temperatures within months (P<0.05). Climatic events are shown above.65

Figure 5.3: Shelf life (ripening *age 4-7) of Cavendish bananas cv. [ililliams

harvested bimonthly and ripened at74,16,18 or 20"C with 300 pL Ll


bananas each; the bar denotes least significant differences between

temperatures within months (P<0.05). Climatic events are shown above.67

Figure 5.4: Dscolouration index of Cavendish bananas cv. Williams harvested

bimonthly and ripened at 74, 16, 1 8 or 20"C with 300 pL L 1 ethylene

where, (0) represents healtþ or yellow peel, (1) slight yellow grey peel, (2)

red/brown vascular sfieaking along banana neck or (3) red/brown vascular

streaking along length of peel. Each column represents the mean of 3

replicates consisting of 6 bananas each; the bar denotes least significant

differences between temperatures within months (P<0.05). Climatic events

are shown above. ... ......69

Figure 5.5: Pulp firmness of Cavendish banana pulp cv. Williams at ripening

stage 6, harvested bimonthly and ripened at14,16,18 or 20"C with 300 FL

Ll ethylene. Each column represents the mean of 3 replicates consisting of

6 bananas each; the bar denotes least significant differences between

temperatures within harvest months e<0.05). -. -....70

Figure 5.6: Soluble solid concentration of Cavendish banana pulp cv. Williams

at ripening stage 6, harvested bimonthly and ripened aI 14, 76, I 8 or 20'C

\Mith 300 pL Lr ethylene. Each column represents the mean of 3 replicates

consisting of 6 bananas each; the bar denotes least significant differences

between temperatures within harvest months (P<0.05). - ....11

List ofFþures

Figure 5.7: Principal component analysis chemical nose score plot tlf

Cavendish bananas cv. Williams ripened atI4,16,18 or 20'C in January

(J), March (M), May(MY), July (JU), September (S) orNovember (N)'

Each data point represents means of banana samples for a single replicate.

Ripeningtreatmentsconsistedof 3 replicates..... -...'..-- -..73

Figure 5.8: Principal component analysis chemical nose loading plot of ions

(variables) derived from Cavendish bananas cv. [4/illioms ripened at 14,16,

18 or 20'C in January, March, May, July, September or November. -. -....74

Figure 5.9: Green life (ripening stage 14) of Cavendish bananas cv. Williams at

ripening stage 6, harvested bimonthly and ripened at 16oC with 50, 300, or

1000 pL Lr ethylene. Each column represents the mean of 3 replicates

consisting of 6 bananas each; the bar denotes similarities between ethylene

concentrations within months (P<0.05). Climatic events are shown

above. -. -..76

Figure 5. l0: Shelf life (ripening stage 4-7) of Cavendish bananas cv. Wil liams

at ripening stage 6, harvested bimonthly and ripened at 16'C with 50, 300,

or 1000 VLLI ethylene. Each column represents the mean of 3 replicates


concentrations within months (P<0 05). Climatic events are shown above.

' ""'77


bimonthly and ripened at l6"C with 50, 300 or 1000 pL L I ethylene

where, (0) represents healthy or yellow peel, (l) slight yellow grey peel, (2)

red/blown vascular streaking along banana neck or (3) rcd/brown vascular

sfeaking along length of peel. Each column represents the mean of 3

replicates consisting of 6 bananas each, the bm denotes similarities

vl

Lnt ofFþures

between ethylene concenfations within months (P<0.05). Climatic events

are shown above. .. - - - -...78

Figure 5.12: Pulp firmness of Cavendish banana pulp cv. Williams at ripening

stage 6, harvested bimonthly and ripened at 16"C with 50, 300, or 1000 pL

L1 ethylene. Each column represents the mean of 3 replicates consisting of

6 bananas each; the bar denotes least significant differences between

ethylene concentrations within months (P<0.05). . ... - - - - -...79

Figure 5.13: Soluble solid concentrations of Cavendish banana pulp cv.

Williams at ripening stage 6,harvested bimonthly and ripened at 16"C with

50, 300, or 1000 ,rLLr ethylene. Each column represents the mean of 3

replicates consisting of 6 bananas each; the bar denotes least significant

differences between ethylene concentrations within months (P<0.05) . . . ..80

Figure 5.14: Principal component analysis chemical nose score plot of

Cavendish bananas cv. Williams ripened at 16"C with 50 (5), 300 (3) or

1000 (l) pL Lr ethylene gas in January (J), March (M), May (NIY), July

(JU), September (S) or November (IÐ. Each data point represents means

of banana samples for a single replicate. Ripening treaÍnents consisted of

3 replicates ... ......82

Figure 6.1 : Brown discolouration observed on nitrogen üeated Cavendish

bananas cv. Williams increased with exposure periods; (A) att ripened, @)

6, (C) 12 and (D) 24 hrs of nitrogen exposure . . . . . . .. .95

Figure 6.2zThe score plot of ripe Cavendish bananas cv. Willioms exposed to

nifiogen aünospheres for 0 hrs (air) at stage 3 or 4 (4, g, - ) at stage 3 for

6, l2,24hrs (C, D, E,- -) ot at stage 4 lor 6,72 or 24hrs (F, G, If ')

.. ... .....97

List ofFþures

Figure 6.3: The loadings plot of ions (variables) derived from ripe, nitogen

exposed Cavendish bananas cv.Williams at ripening stage 3 or 4 for 0, 6,

12 or 24 hrs. .. . ... ... ......98

Figure 6.4: Ascore plot of ripe Cavendish banæras cv. Williams exposed to

(A -)

nitogen for 0 hrs (air), (8, -)

nifiogen for 24 hrs in a low

humidity or (C, -)

nifrogen for 24 hrs in a high humidity. . . ...100

Figure 6.5: The loadings plot of ions (variables) for ripe Cavendish bananas cv.

Williams exposedto nitrogen for 0 hrs (aÐ,24 hrs in a lowhumidity or for

24Iusin a high humidity. ....101

Figure T.lzDiagran of air evacuation set up, consisting of (A) 10-L glass

desiccator, @) vacuum pump, (C ) measuring cylinder and (D) Funnel

(covering a single banana). ....111

Figure 7.2: Vacuun (A) infilfiation of ethanol atnosphere generated from I

mL absolute ethanol (B) in a 10-L glass desiccator (C ) into 6 ripening

initiated bananas (D), in the presence of 160 g Ca(OÐ2 @) . , .. . ..113

Figure 8.1: Cavendish banana (cv. Williøms) appearance on day I of the l-

MCP experiment. Bananas were initiated \4rith 300 þL L-l ethylene gas for

48 hrs at2}'Candthen exposedto l-MCP for (A) 0 (air), @) 3 oLUt, (C )

300 nL Lt or @) 30,000 nL L-r . . . ....131

Figure 8.2: The chemical nose score plot of volatiles produced by Cavendish

bananas cv. Williams held for 24,48 or 72hrs in 3 rú- L-t I-MCP (4,- ),

300 nL L 1 l-tr¡Cp (8, - ) or in air (C,- ) after ripening

initiation. . ... .....133

Figure 8.3: The loadings plot of ions (variables) for ripe Cavendish bananas cv.

Williams exposed to 3 nL L 1 I-MCP, 300 nL L 1 I'MCP or held in air after

ripeninginitiation for24,48or72hrsin. ...-134

vlll

List of Tables

List of Tables

Number Page

Table 2.1:The standard colour guide of Cavendish banana ripening stages

(csrRo, 1972). . ... ... ...8

Table 3.1: Discolouration score chart. . -...'.37

Table 4.1: Ctrlorophyll fluorescence measurements on Cavendish bananas cv.

Williams recorded daily during chilling exposure treatment at 5oC for 5 days

and ripeningat2}"C at ripening stage I .. ...51

Table 4.2zThe daily change in chlorophyll fluorescence parameters measured

on Cavendish bananas cv. William.s that were stored at 5oC for 5 days followed

by storage at22'C until ripening stage 4, or stored at room temperature only

(22'C) until ripening stage 4 (more yellow than green) .. . -. .. ' .. -.52

Table 4.3: The polyphenoloxidase activity in the vascular tissue from the peel

of Cavendish bananas cv. Williams stored at ripening stagel at 5'C for 4 days,

compared to bananas stored at22"C for 1 day at ripening stagel . ... '...53

Table 6.1: Assessments of Cavendish bananas cv. Williams at ripening stage 6,

after expostue to a nitrogen atmosphere for 0, 6, 72 or 24 hrs at ripening stage 3

or 4 at22"C. .........--94


after being ripened at ripening stage 4 in air or nifiogen atmospheres for 24 hrs

with a beaker of water or without a beaker of water at22"C. . ...99

Table 7.1: Ripe characteristics of Cavendish bananas cv. Williams after

ripening with ethylene (300 pL L1) at l8oC, treated by vacuum infilfiation over

air or ethanol and held in the resulting atmosphere for 10 min. or 3 hrs. After

lX

List of Tables

the holding period the bananas were held at l8"C until they reached stage 4 and

then were ripened at22"C turtil assessed. . . . . .. . -. .116

Table 7 .2: Peel and pulp tissue ethanol concentrations of Cavendish bananas cv.

williams after ripening with ethylene (300 pL L 1¡ at 18"C, fteated by vacuum

infiltration over air or ethanol and held in the resulting atmosphere for 10 min.

or 3 hrs. After the holding period, bananas were held at l8"C until they reached

stage 4 ærdripened at22"Cwrtil assessed..... .."'118

Table 8.1: SheHlife of Cavendish bananas cv. Williams following ripening at

20"C with 300 pL L1 ethylene gas for 48 hrs then subsequently exposed to 0, 3

or300nLL1 1-MCP for24,48or72hrsat20'C -..'.....127

Table 8.2: Green life of Cavendish bananas cv. Williams following ripening at

20"C with 300 pL Ll ethylene gas for 48 hrs then subsequently exposed to 0, 3

or300nLLr 1-MCP for24,48or72hrsat20'C ........'128

Table 8.3: Pulp fimtness of Cavendish bananas cv. Williams at ripening stage 6

following rþening at 20'C with 300 pL Ll ethylene gas for 48 hrs then

subsequently exposed to 0, 3 or 300 nL Lr I-MCP for 24,48 or 72fus at

20"c. ...128

Table 8.4: Soluble solid concenfiation of Cavendish bananas cv. Williams at

ripening stage 6 following ripening at2)"Cwith 300 tLLLt ethylene gas for 48

hrs then subsequently exposed to 0, 3 or 300 nL L1 1-MCP for 24,48 or 72 hrs

at2o"c' " """129

Table 8.5: Weight loss (7o) of Cavendish bananas cv. Williams from rlpening

stage 1 to 6 following ripening at2lc\À/ith 300 pLL' ethylene gas for 48 hrs

then subsequently exposed to 0, 3 or 300 nL L-t I-MCP for 24, 48 or 72hts at

20"c. ...129

X

Acknowledgments

Acknowledgments

This research was made possible thanks to an ARC SPIRT grant rn associatron

with Chiquita Brands South Pacific (Australia). The project would not have

been possible without the help and support of the following people:

I would like to thank my supervisors Dr Andreas Klieber, Mr Robert (Bob)

Barrett and Professor Margaret Sedgley for the opportunity to work on this

fantastic project and for their continual guidance and encouragement.

I'd also like to sincerelythank Chiquita Brands South Pacific (Australia)

especially, Mr John Evans, Mr Ted Johnson, Mr Ben Franklin, Mr Bill Chartres,

Mr Joe Trimboli, Mr Ian Trimboli, Mr Adam Gallarello and all the staff at the

Pooraka market Chiquita warehouse (Adelaide), for their enthusiasm, advice

and dedication to the improvement of banana quality. Thanks also to all staffat

the Tully, north Queensland plantation (especially Neville) for their assistance

in providing fruit throughout this research.

Thanks to Dr Graham Jones and Dr Michelle Wirthensohn for their technical

advice and help with the chemical nose. A special thanks also to Mr Adrian

Dahlenburg, Mr Michael Rettke, Mrs Maria Nechvoglod and all other members

of the SARDIpostharvest goup for their continual support, technical advice

and loan of the twist-tester. Furthermore, thanks are also expressed to Dr Edna

Pesis, Dr Amanda Able, Mr Andrew Macnish and Michelle Lorimer for their

advice on thcir specific fields of expertise.

I would also like to express my gratitude towards all the staffand students in

the Horticulture, Oenology and Viticulture Deparhnent of Adelaide University,

xl

Acknowledgments

especially, Kerry Porter, Margaret Muchui, Dr Toby Knight, Ermimnawati

Wuryatuno, Dr Mayuree Krajayklang, Darren Alexandra, Zwanyadza Soroti,

Viswanath Pinapati, Brad llarvey (the past and present members of the

postharvest laboratory).

A special thanks to my wonderfül friends, Joanne 'Hippy Chick', Rosa,

Belinda, Franca, Steve, Helen, Toby, Learure, Joyce, Tony, Ho|ly, Mary, Lisa,

Kerry,, Veronica, Lizandall the grls atthe VirgrniaRamsNetball clubwho

saved me many a time from goin' bananas. I'd also like to thank Guido for

taking a chance and for gr"i"g me the inspiration to finish this thesis.

Finally I'd like to thank my loving family especially; Nonna Francesca, Mum,

Dad, my sisters; Franca artd Joatrna and brother in-laws, Paul and Greg (ory)

and nephews and niece, Dean, Thomas and Alexandra.

Thanks for putting up with me through the good, bad and indifferent times!

This thesis is dedicated to Christina 'Chrissi' Musolino, thânk you for watching

over me.

xü

Abbreviations

a

AA

1.MCP

ACC

AdTI

ANOVA

Area

b

CA

Ca(otr)2

COz

CSIRO

cv.

Abbreviations

l- methylcyclopropene

radius of twist tester blade

acetaldehyde

I -aminocyclopropene-l -carboxylic acid

alcohol dehydrogenase

anaþis of variance

the total size of the elecfion acceptors on the

reducirtg side of photosystem II

width of twist tester blade

controlled atmosphere

calcium hyd'roúde

carbon dioxide

Commonwealth Scientifi c and Industrial

Research Organisation

cultivar

degrees Celsius

day

Denver

oc

d

DE

xltl

Abbreviations

Dept.

DOPA

et oL

EtOH

FID.GC

FIM

Fn

Fo

F¡

Fv

FvÆm

Department

dihydroxyphenylalanine

et alia

ethanol

flame ionisation detector- gas chromatography

fl uorescence induction monitor

the maximum fluorescence level

the level at which fluorescence begins to rise

the fluorescence rate

the variable fluorescence level

the level of effrciency with which the energised

electrons can be transferred through the electron

transport chain

gfam

gram of fresh weight

hour/ hours

Hewlett Packa¡d

internal diameter

Incorporated

kilograms

g. f. wt

hr/ hrs

(tb

HP

Inc.

kg

id

xlv

Abbreviatiors

K*

tûn

kPa

L

KNO3

MA

min.

InL

mm

mM

MV

mlz

N,

nL

nmoles

binding constant

kilomefes

potassium nitate

kilo Pascals

litres

least signifi cant differences

limited

metre

molar or maximum moment produced by amr of

twist tester

modified atmosphere

minute

millilitre

millimetre

millimolar

millivolts

mass to charge ratio

nitrogen

nanolitres

nanomoles

LSD

Ltd.

h

M

xv

Abbreviatiors

NSW

NZ

Oz

OD

Pa

PCA

trrefs. comm.

pH

New SouthWales

New Zealand

oxygen

outer diameter

Pascals

principal component analysis

personal commrurication

A qmbol denoting the concentration of hydrogen

ions in a solution

pollphenoloxidase

photosystem II

non-significance level ofover 5%o

significance level of 5%o

electron acceptor site

Queensland

registered fademark

randomised complete block design

relative humidity

Royal Horticultural Society

reverse osmosis

PPO

PSII

P{.0s

P<0.05

QLD

RCBD

RHS

Q¿.

@

RH

RO

xvl

Abbreviatiors

SDS

SSC

Tf max

USA

WA

wlv

pmol

vlv

sodium dodecyl sulphate

soluble solid concentration

the time frame at which the maximum

fluorescence occurred

United States of America

volume to volume

Washington

weight to volume

microlitres

micromoles

percent

crushing sfrength

maximum arm angle before tissue failure

pL

otfo

6

e

xvll

Clreptec I

1. Getteral Introduction

Bananas are transported to most markets green in their pre-climacteric state and

are then ripened according to market demand. The ripening process of

transforming the physiologrcally mature, but inedible green fruit into a visually

appealing sweet tasting product (Wills et a1.,1998) is a very important stage in

the overall marketability of bananas.

The marketability of bananas is detennined by a number of consumer

preferences. Visual appearance of food is a major influence in consumer

acceptance (Francis, 1977) and bananas are no exception, with peel

discolouration, caused by enzymatic browning, being the main consumer

concern affectingbanana acceptability and causing a significant impact on fruit

marketability (Omuaru and Izonfuo, 1990). Another consumer concern is the

speed of banana deterioration. The short shelf life of bananas can be as low as

onlythree days (8. Chartres, ChiquitaBrands SouthPacific, pers. comm',

1999). Research into the buying habits of people has concluded consumers

would purchase more bananas if they had longer shelf lives (Ausfralian Banana

Grower's Council, 1999). These main concems in the banana industry

emphasize the need for research into the pre-climacteric, climacteric and post-

climacteric stages of postharvest life, to improve the shelf life and quality of

ripening bananas.

1

Chapter 1 : General Introduction

Banana appearance is negatively affected bypre-climacteric chilling in the field

or during transpott, and early detection of chilling ittj.t y synptoms that become

visible after the onset of ripening would allow marketers to re-grade and re-

direct affected fruit to appropriate altemative markets.

Uniform ripening can be accomplished by controlling temperature, ethylene

and humidity (Scott and GandanegaÍa,197 ;Peacock, 1980; Kim and Lee,

1988; Wills , L990; Seberry and llarris, 1993). However, banana quality does

varythroughout the year @. Chartres, ChiquitaBrands South Pacific, pers.

comm., lggg),bUt little research has focussed on optimising commercial

ripening protocols párticularly in relation to banana ripening temperatures,

ethylene concentratidns and different harvesting times of the year.

Most postharvest research has been performed to extend green life of pre-

climacteric bananas to transport them to distant mafkets. For example,

postharvest treatments such as controlled or modified atmosphere storage

(Yahia, 1998), nitrogen ahrosphere storage (Wills et a|.,1990) andthe ethylene

antagonist l-methylcyclopropene (l-MCP) (Golding et a1.,I998;Macrrjsh et al.,

1998) have all been reported to slow down the onset of the ripening process in

bananas. Few studies have focussed on extending the shelf life of eating ripe

fruit and often have not investigated the effect on banana quality thoroughly.

To ensure improvedmarketing of bananas with better consumer characteristics

in this highly competitive industry, frirther research is required into the

posthawest management of bananas. The main aim of this research was to

extend the posthawest life of bananas without compromising banana

appearance or eating quahty. Screening techniques of chlorophyll fluorescence

and polyphenoloxidase activity measures were examined for early detection of

2

Chapter I : General Introduction

chilling injury. Ripening and postharvest procedures for Cavendish bananas cv

l4lilliams were also studied throughout the year by applying different

concentrations of ethylene and various ripening temperatres to optimise

industryripening procedures. Furthermore, the use of nitrogen atmospheres,

ethanol atrnospheres and l-methylcyclopropene (l-MCP) during the post-

climacteric stage were examined to extend banana shelf life while maintaining

visual and eating quality.

J

Chnprer 2

2. Literrture Review

2.I Banana classiflcation

Bananas along with grapes, oranges and apples are fruit that are easily

recognised in countries around the world. The banana belongs tothe Musaceae

family and is gIo\MIt in many tropical and subtropical regions world wide (Johns

and Stevenson , 1979 Salunldre and Desai, 1984). A popular edible banana that

has been derived from wild ancestors is the Cavendish cultivæ (Johns and

Stevenson, lgTg).Bananas are classified as a berry fruit. The commercially

grov,nMusa cavendishii is a six sided, seedless, angular berry that ripens from a

green to a bold yellow colour.

Cavendish is the main cultivar used for world trade (Seymour, 1993, Wills e/

al.,\998) and'Williams' is the principal clone grown in Australia, contributing

to 95%o of all bananas harvested @aniells, 1986). Harvesting usually takes

place when the fruit is 7 5%o mahre and has ¿ hard and green appearance.

During the postharvest period, the fruit becofnes soft, edible, yellow and

aromatic.

4

Chapter 2: Literature Revrew

2.2 Climacteric fruit

Fruit that are able to produce ethylene autocatalytically after harvest are

classified as climacteric fruit (Tucker, 1993). Climacteric fruit, such as

bananas, have a sizeable increase in their respiration rate from 20 mgCO2 kgl

hr-l prior to ripening initiation to a climacteric peak of 125 mg CO2 kg1 hr-l. As

ripening progresses the respiration rate reduces again to about 100 mg CO2 k91

hr-l lPalmer ,l97l; Salunkhe and Desai, 1934). However, the respiration rate in

bananas increases only after endogenous ethylene production increases from

0.05 to 3 ¡rL ethylene kgr hr-l (Se¡nnour, 1993). This increase in ethylene

production after harvest coincides with physiological mahrration and the onset

of ripening. hritiation of the ripening phase in bananas begins commercially

with the exogenous application of ethylene to fruit attheT5o/omature, green

stage (Taiz and Zeiger, I 998).

Climacteric and non climacteric fruit will both respond to exogenous ethylene

with an increased respiration rate, but non climacteric fruit will respond only

proportionally to the level of exogenous ethylene applied and respiration rate

will decrease as soon as they are removed from the ethylene enhanced

atmosphere (Wills et al., 1998). Therefore, only climacteric fruit produce

ethylene autocatalytically, and this is triggered commercially by the exogenous

application of ethylene to bananas, ensuring that all fruit ripen uniformly when

required.

2.3 The ripening process of bananas

The process of ripeningbananas involves physiological, biochemical and

physical changes to fruit (Wills, 1990). Ethylene is a plant hormone that

initiates the ripening process of bananas (Sjaifullah and Dasuki , 1992 Taiz and

5


Zeiger,1998). While mature fruit are attached to the plant, the sensitivity to

exogenous ethylene is inhibited by other plant growth regulators such as auxins,

although their exact regulatory role is unclear. However, ftuit that me removed

from the plant become ethylene sensitive soon after harvest (Stover and

Simmonds, 1987; Septour, 1993).

Ethylene sensitivity may be a problem when fruit æe in transit, especially

travelling interstate or internationally. To delay the effect of any ethylene

contamination that mây oçcur, batranas are transported at l3"C (8. Chaltres and

T. Johnson, Chiquita Bmtrds South Pacific, pers. comm .,1999-2000). The low

temperature (13'C) reduçes fruit metabolism and prevents ripeqing from

occurring in bananas (Wills et al., 1998). Upon arrival at the intended market,

the application of high (>300 ¡rL Lt) levels of ethylene at temperatures between

l4-20'Cto induce ripening are common among commercial distributors (8.

Chartres and T. Johnson, Chiquita Brands South Pacific, pers. comm -,1999-

2000).

The biochemical breakdown of starch and changes to pulp cell wall structure

bypectin degrading enzynes decreases the cellular rigdity of banana tissue and

therefore physically softens the pulp (Seymour, 1993; Thompson, 1996). Other

non pectic polysaccharide degrading enrymes like cellulase have also been

identified in ripening bananas, which may also contribute to the softening of the

pulp (Palmer,l97l; Seymour, 1993). These processes may occur independentþ

of each other, but to ripen a fruit uniformly and correctly the initiation of each

process must occur (Thompson, 1996).

The most identifiable difference that consumers associate with unripe and ripe

fruit is the peel colour change that occurs during the ripening of bananas. The

6

Cløpter 2: Literature Revrew

CSIRO banana ripening chart (1972) used by most commercial businesses,

shows bananas converting from green fruit into yellow fruit with brown areas, in

atotal of eightnpening stages (Table 2.1). Bananasbetweenripening stage 4

(more yellow than greÐ to s{age 7 (yellow with some brown flecks) are

preferred by 96% of consumers (Anonymous, Your guide to geater profits,

undated).

7

Chapter 2: Literature Revrcw

Tabte 2.Lz The standard colour guide of Cavendish banana ripening stages

(cslRo, 1972).

Stage

Peel colour Approximateo/o slarchcontent

Approximate %sugar conten!

Comments

I Green

SPRUNG Green

20

19.5

18

76

13

2.5

1.5

1.0

0.5

1.0

25

4.5

7.5

13.5

18

t9

t9

Hard, rigid;no ripening

Bends slightly,ripening started

Peels readily;firm ripe

Fully ripe;aromatic

Over-ripe; pulpvery soft anddarkening, highlyaromatic

2

J

4

75

6

7

Green, trace

of yellow

More green

than yellow

More yellowthan green

Yellow,green tþFull yellow

Yellow,lightlyflecked withbrown

Yellow withincreasingbrownflecks

8

8

Cl:øptr' 2: Literatwe Revrew

Coloured pigments that are found in bananas throughout their various stages of

development include chlorophylls (green), carotenoids, xanthophylls (yellow)

andmelanins (brown) (Stover and Simmonds, 1987; Thompson, 1996). A

change in peel colour during ripening occurs due to the degradation of

chlorophylls, even though there is a constant carotenoid level throughout the

banana ripening stages (Thompson ,1996).

The appearance of unsightlypigments or the failure of yellowpigments being

revealed at specific stages in banana development can occur, if adequate

ripening conditions are not provided (Thompson,7996). Problems associated

with inadequate ripening conditions include the delay of banana ripening, lack

of taste and flavour on ripening, peel colour changes such as the red-brown

discolouration of fruit that have been affected by low temperature sfesses, or

the failure of fruit peel to ripen due to high temperature stresses Qvlwata,1969;

Moris, 1982; Torasker and Modi, 1984; Thompson, 1996; Wills e/ a|.,1998).

Various changes to banana volatile components also occur during the ripening

process. Major volatile components of green bananas are hexanal and tans-2-

hexenal (I\4arriott, 1930). Alcohols and carbonyls give the gieen dnd woody

aroma of unripe bananas, whereas esters make up the main fraction (70%) of

ripe banana ¿ìroma (Tressl and Jennings,l972;Marriott, 1980; Stover and

Simmonds, 1987; Seyrnour, 1993). Although the exact composition of esters,

alcohols, ketones, aldehydes and phenol esters making up the aroma and flavour

of bananas is cunentþ unclear (Seymour, 1993), it is known that the 'banana

like' ærd 'fruity' aromas are associated with the amyl esters and butyl esters,

respectively (Tressl and Jennings,l972;Marriott, 1980). Volatile production

increases during ripening, but levels off and decreases during senescence

9

Chapter 2: Literature Reuew

(ripening stage7 and 8)

Aroma constituents are different at the various stages of ripening depending on

ripening temperatures (Marriott, 1980). Therefore, the analysis of volatile

constituents to examine the effect of various ripening conditions on the aroma

of ripe bananas will be useful in determining undesirable aroma differences

between bananas growTì at various times of the year and ripened using different

ripening procedures.

Temperature, ethylene and humidity levels are tlree factors regulated during

ripening of bananas and these are believedto have a determining effect on final

product quality and storage life (Peacock, 1980; Paull, 1993; Wills et a1.,1998);

therefore knowing the optimal levels of these factors will also be beneficial for

increasing product quality

2.4 Temperature in relation to ripening

Extreme temperatures (<13"C or >26"C) are a critical factor that influences the

quality of the final ripened product (Thompson,1996 Turner, 1997;Wills et

al.,1998). Temperature control of banana fruit while in the field is diffrcult and

therefore differences in field temperatures between winter and summer seasons

occur. This field temperafure difference leads to visual appearance differences

between bananas (Turner, 1997), although the severity of visual appearance in

relation to the commercial ripening procedures used after harvest has not been

explored.

Unlike in the field, temperatures of fruit after harvest can be controlled.

Regulating temperature is the principal way to confiol respiration rate, ripening

and senescence. As temperahres drop, so too do reqpiration rates in produce

l0


which increases theirpostharvest life (Paull,1993). In addition lowering of

storage temperatures decreases fungal growth, tissue softening and water loss

(Patterson, 1980). Ripening temperature experiments performed on Cavendish

bananas found that temperatures between 13.9 and 32.2"C do not affect the

eating quahty of bananas (Peacock, 1980), but importantly shelf life decreased

exponentially with increasing temperatures. Skin colour was found to be dull

(yelloWgrey) in appearance and without total chlorophyll degradation at stage 6

at26.7'C or above. This research therefore suggests that only a nalrow

temperature range (14-24"C) would be suitable for the ripening of Cavendish

barianas.

2.4.1 Low tpmperatures

Even though most produce benefits ftom storage at low tempetatures, topical

and subtropical fruit react adversély to temperatures between 0 dnd 13"C

(Maniott, 1980; Israeli and I^ahav, 1986; Wills et a\.,1998) Bananas in

particular show the negative effects of low temperatrue at l2"C and below

(Wills et aI.,1998). This temperature stress causes the physiologrcal disorder

chilling injury, which is not only a postharvest problem, but can also occur in

plants throughout their development inclucling in the field, during üansport, as

well as in storage (Saltveit and Morris, 1990; Kays, 1991; Kays, 1999). Couey

(1932) reported that the sensitivity of ripe bananas to chilling ittjury is less than

that of unripe bananas. Therefore, lower temperatures could possibly be used to

store ripe bananas to extend banana shelf life without compromising banana

quality.

Temperatures of l2oC and below cause a rapid primary response tn unnpe

bananas. The primary response is a physical change in membrane lipids

11

Chapter 2: Literatwe Revrew

including ion movements and protoplasmic sfreaming and a dissociation of

en4lmes or proteins (Wills et al., 1998). This response is reversible if the

temperature returns above the critical temperature of l2"C before secondary

responses like membrane breakdown occur (Saltveit and Morris, 1990; Wills ef

at.,I998). If temperahres continue to remain below 72"C to the point where

secondary responses occur, then irreversible injury will become evident (Figure

2.1).

t2

Chapter 2: Literatwe Revrew

Time below Iz"C

Primarv Response

Reversible

+ Imrnediate response

= Physical membrane changes

* Protein and lipid changes

Secondarv Resnonses

= Cellular membrane integnty disrupted

* Reduction in protoplasnic sh'earning

* Recluction in respiratíon and protein syrthesis

+ Decreases ion movements tlu'ough membranes

x Disruption of enzymes and rnetabolic

processes

+ Iæads to appearance of visible syrnptoms

* Browning in vascular tissne

* DulVyellow/grey peel appearance

* Loss offlavour

Necrosrs death

lïgure 2.lzBananaresponses to temperatures below l2"C thatlead to the

appeararìce of chilling injury on fruit (after Wills et al.,1998).

Irreversible

t3


At the irreversible stage, secondary responses in the form of visible chilling

injury synptoms occur. These s)..rnptoms include subepidennal bror¡¡n

streaking in the vasculm tissue of the fruit, loss of flavour, delayed ripening,

hardening of the cental placenta, a build up of tannins, skin discolouration,

slow starch to sugar conversion, decrease in ascorbic acid level, watery dark

green patches on the skin and brittleness of the banana fingers @antastico et al.,

1975; Morris, 1982; Paull, 1993).

Many of these symptoms negatively affect the visual appearance of the bananas

and as the appearance of the fruit is a major criterion for consumer buying,

chilling injury of bananas is of major concern. Chilling injury is very easy to

observe on bananas, as they are uzually a dull grey yellow colotn or in severe

circumstances, show large red-brown discoloured areâs. However, these

symptoms may not appear until the fruit is subjected to non chilling conditions

(Saltveit and Morris, I 990).

2.4. IJ Chlorophyll tluorescence

Changes in chlorophyll levels in banana peels usilrg fluorescence induction,

may offer a means of detecting damage caused by environmental stresses such

as low temperatures before visual s5imptoms appear @eEll et al. , 1999).

Chlorophyll fluorescence induction is an easy, quick and non destructive

measurement that can be perfonned to measure various chlorophyll

fluorescence pararneters including:

o Fo - the level at which fluorescence begins to rise

o Fm - the maximum fluorescence level

14

Chapter 2: Literature Review

o Fv - the variable fluorescence level

o FvlFm - the level of efficiency with which the energised electrons can be

transferred through the electron fansport chain

o Tf max - the time frame at which the maximum fluorescence occurred

o Area - the total size of the electron acceptors on the reducing side of

photosystem tr (PS tr)

@eEll et a1.,L999).

The reasoning behind using chlorophyll fluorescence as a measure of this

environmental stress on plants, is that functional properties of the chloroplast

thylakoid membranes change during chilling injury, which influences the

emission of chlorophyll fluorescence (Smillie, 1979; Smillie and Hetherington,

1990). Fluorescence induction occurs because chlorophyll fluorescence mirrors

the processes of photosynthesis in the chloroplast of the plants, including light

absorption by carotenoids and the photosynthetic energ¡'transfer in photosystem

tr (PS tr) that results in a release of fluorescence emission (Goodwin, 1980;

DeEll et al.,\999). The fluorescence measurement takes place in PS tr.

Fluorescence is induced by illumination of the plant material with red light.

The primary reaction site of the fluorescence is believed to be at the electron

acceptor site (QÐ of PS tr, where a rise in induced fluorescence directly relates

to the reduction of the QA in PS tr (Smillie, 1979, Smillie and Hetherington,

r ee0)

Fluorescence emission from chlorophyll a is susceptible to disruption due to

cellular injury caused by low temperature stress (Smillie and Hetherington,

15

Chapter 2: Literature Reuew

1990). Chilling injury causes inhibition to develop on the photo-oxidising side

ofPS tr and so reduces the induced chlorophyll fluorescence, whereas plant

tissue that had not previously been chilled would inhibit the photoryarthetic

electron transfer on the photo-reducing side of PS tr increasing the yreld of

fluorescence (DeEll et al.,1999). This means there would be an expected

difference in chlorophyll fluorescence emission between cell-disrupted, chilled

fruit and cell-intact, non-chilled fruit.

However, there has been very little research in the determination of the useful

chlorophyll fluorescence parameters for chilling injry analysis. The

chlorophyll fluorescent parameter presumed to be of most value when

measuring membrane altering damage like the darnage caused by chilling injury

is the FvlFm parameter, because it can be measured within one second (DeF,ll et

a1.,1999). A recent study @eEll et a1.,1999) reported that bananas stored

between 5'C and l5'C showed a decreasingFvÆo ratio, similar to that of

conftol bananas during ripening as chlorophyll degades. This finding suggests

that perhaps the Fv and Fo parameters may not be very useful in determining

chilled bananas from non chilled bananas, contrary to a report by Jacobi et al.

(1998) who found the Fo measurement was very important in determining

abnonnal skin ripening in mangoes. Therefore, further study of this method of

analysis is required to observe the effectiveness ofusing chlorophyll

fluorescence parameters as an early chilling rnjury detection method on mature,

green banana peel.

2. 4. 1. 2 Polyp heno loxidas e activily in c hilled frait

Another possible early measurement of chilling injury in Cavendish bananas

may be the analysis of polyphenoloxidase ePO) activity present in chilled

16

Cløpter 2: Literature Rwrew

Cavendish bananas, compared to the PPO activity in non chilled Cavendish

bananas. Polyphenoloxidase activity in the peel of bananas, is reported to be an

indication of browning, which is a symptom of chilling injury (Murata 1969).

Two types of reactions can result from the activity of the copper containtng

enz¡ime PPO:

l) Monophenolase - the hydroxylation of monophenols to odiphenols and

2) Diphenolase - the dehydroxylation of monophenols to o-quinones

Bananas undergo only the second reaction producing o-quinones. These o-

quinones then undergo a sequence ofpol¡rnerisation reactions that produce the

brown pigments characteristically found in chilled fruit (Zawistowsl<t et al.,

199 I ; Whitaker and læe, I 995). This reaction can be achieved by PPO as well

as other enz¡nnes like peroxidase (Kalyanar¿ünan et a1.,1984).

In healthy fruit cells, PPO is found in plastids, whereas phenolic compounds are

located in the vacuoles. However, upon cellular disruption the plastid and

vacuole contents are able to combine, resulting in an enrymatic reaction that

leads to brorvning. PPO activity in bananas was found with gamma irradiation

to conelate with peel and pulp browning, suggesting conforrnational changes

increased PPO activity (Amiot et a1.,I997). Therefore it is probable that

cellular disruption caused by low temperatures will increase PPO activity.

The oxidation of phenolic compounds such as dihydroxlphenylalanine (DOPA)

occurs due to PPOs in the presence of oxygen (Figure 2.2) (Jones et a1.,1978;

Eskin, 1990; Amiot et a1.,1997). As aresult of low temperatures breaking

down cellular structures, the en-ryme and substrate are able to interact to

t7


produce unstable quinones. Low temperatures also encourage the conversion of

quinones to phenols by ascorbic acid, rather than following alternative pathways

(Abd El-Wahab and Nawwar ,1977). Those phenols are then able to undergo

en-rymatic reactions that lead to an accumulation of quinones and

polyrnerisation reactions forming the brown discolouration in chilled fruit.

OH

HO

FA,SÏ+*rçoCFh

N

D OPAMIHE

MELA}IIN(ûEHEEAL

ABSOR PTION)

Nl+ rr

23.0IHYDR OIND OLE-5Ë oulN[]HE (RED)

D OPAhrllNEOUINOHE

LOW

o

{-NH H

IND ÛLE-5ÉtrurNDHE (FU ÊPr-E)

5,Ê DIHYDR OXYHD OLE

Figure 2.2:The reaction mechanism for oxidation of dopamine by banana

pollphenoloxidase (Palmer, 1963).

18

Chapter 2: Literature Revrcw

2.4. 1.3 Peroxidase activity

Toraskar and Modi, (1984) found that chilled fruit showed a lower peroxidase

activity than non chilled fruit. Peroxidase is an enzyme that is associated with

ripening processes in fruit. Peroxidase activity does not directly relate to

chilling ioj,rry symptoms, but it is directly related to the ripening process

(Torasker and Modi, I 984; Dilley, L97 0).

In fact, fruit that have been chilled once ripening has been initiated may not

show any significant differences in peroxidase activity compared to non chilled

fruit, but may showphysical signs of chilling injury. This would explain why

Zatberman et al. (1988), found that even though peroxidase activity was

inítially lower in the peel of mango stored at low temperatures, it increased after

chilling injury synptoms began to occur on the ripened fruit even though fruit

remained in low temperatures. Therefore, peroxidase activity is unlikely to

provide a method of early chilling injury detection.

2.4.2 Hightemperature

Temperatures of 25oC and above cause a physiological disorder in Cavendish

bananas known as 'pulpa creme' or 'yellow pulp'. 'Pulpa creme' is the failure

of the banana peel to completely degreen, even when the pulp tissue is at the

soft and edible stage (Thompson, 1996) due to an inhibition in the breakdown of

chlorophyll in the peel. Consequently, ripeningtemperatures below 25oC are

required to ripen bananas for consumer appeal.

2.5 Ethylene in relation to ripening

Ethylene (CzII+) is recognised as an important plant growth regulator of plant

functions and biosynthetic pathways, particularly influencing the ripening

l9


process in ftiit (sjaifullah and Dasuki ,1992;Taiz and zniger, 1998;wills et al.,

1998). Ethylene is recognised as having a number of roles to perform during the

ripeningprocess of climacteric fruit (Rhodes, 1980).

1. Controlling the initiation of ripening.

2. Increasing the internal production of itself as a ripening response.

3. Changes in colour, texture and flavour

4. The promotion of other ripening processes.

Pre-climacteric bananas have a constant endogenous ethylene production level

(0.05 pL ethylene kgt ht-t), although this level increases sharply (3 pL ethylene

kdt ht-t) prior to the respifation rise that occurs at the onset of ripening (Paull,

1993, Seyrnour, 1993). The autocatalytic biosynthesis of ethylene @gure 2.3)

that occurs in pre-climacteric bananas can be initiated by exogenous ethylene

application (Tucker, I 993).

20

Clmpter 2: Literature Revrew

ATP

Methionine S-adenosylmeth ionine (SAM)

ACC synthase

1 -aminocyclopropene- 1 -carboxyl ic acid (ACC)

ACC oxidase

Figure 2.3: Simplified ethylene biosynthetic pathway in ripening fruit (after

Wang, 1989).

Ethylene levels of 100 to 1000 pL Ll for 24hrs, applied either as a single shot

of ethylene gas or in a frickle mechanism, are used for initiating rþening in

climacteric fruit in commercial situations, although levels as low as 0.1 or 1 ¡rI

I,r for 24ltrs are equally as effective in initiating ripening (Seymour, 1993;

Dominguez and Vendrell,l994;Wills e/ a1.,1998). Despite this, the optimum

amount of exogenous ethylene gas to initiate ripening while resulting in the best

quality produce with an extended shelf life is not known.

Scriven et al. (1989) found that fiained panellists detennined distinct flavour

and aroma differences between Cavendish bananas ripened with ethylene

Ethylene

2t

Chapter 2: Literature Review

compafed to those ripened on the plant. The flesh from naturallyripened fruit

was described as being 'more fruity, less green and softer than ethylene treated

fruit.' However, the eating quallty of bananas exposed to lower (<300 pL Lt)

ethylene levels were not compared to those exposed to higher (>300 FI Lt)

ethylene levels. This may be a useful evaluation to observe whether quality

difference can be detected in fruit. If eating quality is affected by the exposure

to different ethylene levels, the industry may choose to regulate the addition of

ethylene more closely.

2.6 Humidity in relation to ripening

The relative humidity (RII) of storage facilities can be adjusted to satisfu fresh

produce requirements. This is essential for bananas, as they are removed from

their constant water supply at harvest. If excessive transpiration in low humidity

is allowed to proceed unchecked, water loss will rezult in banana shrinkage

(Grierson and'Wardowski, I 978).

Akkaravessapong et al. (1992) and Blankenship and Herdeman (1995) have

researched the effect of relative hunidity on banana fruit quality and their

susceptibility to mechanical damage. They found that low relative humidity

(50%) is detrimental to fruit quality, increasing the rate of water loss in bananas

three to four times over bananas stored lrr.90% RH. In fact, Blankenship and

Herdennan (1995) recommend ahighhunidity of 95%RHthroughout the

ripening period. This results in reduced banana peel scarring and so generally

gives a more visually appealing banana compared to those ripened at lower

humidities (<65n.

A disadvantage of storing bananas at a humidity level of 95% is the

22

Ctøpter 2: Literature Revrew

encouragement of the growth of undesirable micro-organisms leading to fruit

decay. Decay occurs mainly when fruit are soft or damaged prior to storage and

accelerates in high humidity storage (Sharkey and Peggie, 1984). Sharkey and

Peggie (1984), however, observed a higher incidence of peeling injury on

peaches (where the skin becomes separated from the flesh of ripe fruit when

hærdled) when stored in high humidity compared to low humidity for 4 weeks.

The thickness of banana peels as opposed to peach peel would reduce this

problem with bananas.

2.7 Post ripening storâge

Higþ ethylene levels and respiratory rates in bananas hasten the ripening

process and reduce storage life (Paull, 1993;TaizandZeiger,1998). Banana

sheHHfe often can be as short as three days, when stored at supermafket

temperatures of 23oC, compared to seven to 28 days when stored atl4oC @aull,

1993; J. Evans, Chiquita Brands South Pacific, pers. comm., 1998). Continual

temperature regulation at l4"C within the superrnarket is unlikeþ. This poses a

dilemma for suppliers wanting to speedup the ripening process while extending

storage life by slowing down senescence. Bananas need to be maintained at the

'ideal' eating stage for as long as possible, to ensure consumer satisfaction and

maximum profit. As 40% of consumers prefer to eat bananas at stage 5 (yellow

with green tips) and 32o/oprefer ripening stage 6 (all yellow) (Anonynous, Your

gUide to greater profits, turdated), post ripening treatments need to be used to

extend banana shelf life for as long as possible.

2.7.L Controlled atmosphere and modified atmosphere storage

Fruit metabolism can be slowed down during postharvest storage by appþing

an atmosphere that has an increased carbon dioxide (5o/o) andlot reduced

z)


oxygen (2Y")levelcompared to normal ar (0.03% CO2 and 2l%Où (Yahia,

1993). This modification of the atrnosphere reduces the banana's sensitivity to

externally applied ethylene and decreases the deterioration of fruits by inhibiting

endogenous ethylene production and consequently prevents a rise in the

respiration rate (McGlasson and Wills, I 972 ; Wills, 1990 ; Kader, 1992;

Elyatem et a1.,1994). Additional benefits are the reduction of the fruit's

softening rate and composiúonal changes, which slow down senescence (Kader,

tee2).

Previous experiments (McGlasson and Wills, 1972; Wills, 1990), have

concentrated on using controlled or modified atmospheres (CA/MA) to delay

banana ripening while in transit. However, storage in controlled or modified

atrnospheres after ripening may also be beneficial for the extension of sheHlife.

According to CO2 and 02 tolerance levels stated by Yahia (1998) and Kader

(1993), green bananas can withstand long periods of storage in2o/o Oz and 5%o

COz at 15'C. However, they did not mention the effect of controlled or

modified atrnosphere storage of bananas applied at post ripening stages, such as

ripening stage 4,the exact duration of storage or the overall effect that CAAvIA

storage has on fruit quality. A disadvantage of MA storage is the need for the

packaging to stay intact throughout storage to maintain the internal aûnosphere,

which would be costþ due to the labotr required to repackage damaged MA

bags during the ripening period.

2.7. 1.1 Nitrogen atmosphere storoge

A cheaper ærd easier mcthod of extending banana shelf life than MA packaging

may be the storage of bananas in nitrogen atmosphere (i.e. ultra-low oxygen, 0-

1.5'/ù promptly after harvest. It would be cheaper and easier, because there

u

Cløpta 2: Literature Review

would be only one application of nitrogen required. Wills et al. (1990), found

that storing bananas in nitogen for three days at 20'C within three days after

harvest (ripening stage l), extended the total time taken to ripen (to stage 5,

yellow with green tips) by eight days. However, fruit that were damaged prior

to being treated with nitrogen tended to have increased peel injury (Wllls et al.,

1990). These findings are an encouraging sign for further research into nitogen

storage in relation to the extension of shelf life.

Importantly, banana storage in nitrogen prior to ripening (stage l) must occur

no later than three days after harvest (Wills et a1.,1990). Treating

preclimacteric fruit with nitogen could occur on-farm prior to ripening, to

ensure long distance shipping of green bananas. However, instead of treating

bananas prior to ripening, flrther research on the effect nitrogen alnospheres

have on post-ripened banana shelf life is required. The storing of bananas at a

post-ripened stage in nitrogen would be a convenient way of handling bananas

with the intention of extending fruit shelf life (from ripening stage 4 to *age 7),

instead of green life.

2.7. 1.2 Ethanol atmosphere storoge

Ethanol (EtOÐ atmosphere storage is another method available to extend

banana sheHlife. The biosynthesis of ethanol occurs naturally during the

senescence phase of banana ripening (Tressl and Jennings,L972;Marriott,

1930). However, endogenous levels of ethanol can also be induced earlier in

the ripening phase by storing fruit tmder anaerobic conditions (Pesis and

Marinansþ, 1993; Pesis,1996). The induction of endogenous ethanol while

under anaerobic conditions is an effective method of inhibiting or delaying the

ripening processes. In fact Podd and Van Staden (1998) reported the inhibition

25

Clmpter 2: Literature Revrew

of ripening by ethanol is associated \Mith a decrease in ethylene production, by

preventing the conversion of ACC to ethylene. These inhibitory effects of

endogenously synthesised ethanol can and have been simulated by exogenousþ

applyrng ethanol (Ratanachinakom et a1.,1999); a volatile that is a natural

ripening product of bananas.

The storage of fruit in an ethanol atrnosphere has been found to delay ripening

of breaker stage tomatoes (Salweit and Sharaf, 1992). Similarly low oxygen

atmospheres that induce an accumulation of ethanol promote anaerobic

metabolism and delay ripening. @esis and Avissar, 1989, Pesis and Marinansþ,

1993; Pesis,1996). Consequently, studies into the application of ethanol

atrnospheres to climacteric fruit is of considerable interest to researchers.

Hewage et al. (1995) and Ritenour et al. (1997) have since reported that the

application of an ethanol atmosphere to bananas prior to or after the climacteric

rise is not effective in extending banana green or shelf life. Ethanol was applied

through ethanol soaked filter paper in a treatment chamber, however, neither

reported endogenous ethanol levels of bananas before or after ethanol treatment.

This would be an important measurement as bananas have thicker peels than

tomatoes and uptake is therefore not ensured (Hong et al., 1995).

Ratanachinakorn et al. (1999) used ethanol vapour vacuum infilnation to force

ethanol into tomatoes which ensured ethanol was absorbed through the tomato

skin and taken up through the fruit calyx. This method may be a useful option

in the effort to drive ethanol into the banana peel and pulp tissue, and to extend

banana shelf life.

2.7.2 Ethylene antagonist

Exposure to ethylene is a common practice in most commercial markets to

26


promote even ripening in bananas. However, even though exogenous ethylene

atmospheres are beneficial for fruit ripening uniformity, it also hastens ripening

and senescence. Recentþthe discovery of l-methylcyclopropene (I-MCP), an

organic molecule that blocks the ethylene receptor preventing ethylene

production, has been identified as an ethylene antagonist with potential use in

the commercial fruit industry.

I-MCP is a planar, strained olefin with a methyl goup adjacent to the double

bond (Figure 2.4) (Sisler et o1.,1996; Sisler,2000). Olefins compete with

ethylene to bind to ethylene receptors, but unlike ethylene, the süained olefins

bind tightly to the receptors preventing an active response (Sisler et a1.,1996;

Sisler and Serek, 1997 ; Jiang et al., 1999b). The ethylene receptor is believed to

contain an unidentified electron donor metal, that olefins bind to. The theory of

why ethylene forms an active response and 1-MCP does not, relates to the

ability of I-MCP to bind tightly to the ethylene receptor (I-MCP was bowrd for

12 days in bananas teated at24"C). This is unlike ethylene, which leaves the

complex after withdrawing its electrons from the metal, causing ligand

substitution that induces an active response @igure 2.5) (Sisler and Serek,

1997). An advantage of 1-MCP is that it seems to be non toxic at active

concentrations (Sisler and Serek, 1997). Currently, I-MCP can be applied to

indoor non-food crops in the USA (Environmental Protection Agency, 7999),

such as fresh cut flowers, and its availability commercially for use on food crops

such as bananas is likely in the future.

27


Cru

1-methylcyÊlopropene

Figure 2.42 T\e structure of I -methylcyclopropene

Inactive

L2

Active

L3

+[+Ç] ----l+ -Dt Lt- kE' l-4

Inactive

L3L1 L1

J=

ü

,,-J, +f

lnactive

L2

+ 1-MtP+h +

lnactive

L¡ L3

t..'L1-¡i4: - --.

1-MCP 14

2q

J-

Figure 2.5: Model of ethylene and I-MCP probable action on the ethylene

receptor. Electron donor metal (M) wrth ligands of unknown structure (L) binds

to ethylene (E) or I-MCP. Ethylene completes its electon fransfer and is

releasedleavingthe resultrng complex active while I-MCP binds so tightlyto

the complex itremains attached resulting in an inactive complex (Sisler and

Serek, 1997).

28

CIøpter 2: Literature Reuew

The anti-ethylene agent I-MCP has been tested on green (stage l) Cavendish

bananas prior to ethylene initiation and has been found to effectively slow

ripening by a factor of four compared to untreated fruit (Golding et al.,1998;

Macnish et a1.,1998;Jianget al.,l999a;Jianget al.,l999b;Harris et al-,2000;

Jiang et a1.,2000;Macnish et a1.,2000b), allowing extended storage of green

bananas. 1-MCP inhibited the production of endogenous ethylene by

suppressing the critical ethylene producing enrymes ACC synthase and ACC

oxidase or by forcing the production of the inactive malonyl ACC (Golding er

aL.,1998).

A nunber of studies has also investigated the effect 1-MCP has on banana

ripening, applied afterripeninginitiation (Macnish et a1.,1997;Goldinget al-,

1998; Goldinget a1.,1999;Jianget al.,l999b; Joyce et a1.,1999;Macnish er

at.,2000a). The studyby Golding et al. (1998) showed the application of l-

MCP prior to or 12 hrs after the application of propylene, a comparable and

effective analogue of ethylene (Rhodes, 1980), affected some biochemical

processes compafed to normally ripened fruit. I-MCP caused the suppression

ofvolatile production, uneven peel colouring, reduced respiration rates, an over-

production of ethylene and an extension in green life. The same studyreported

the application of I-MCP 24hrs after the initiation of ripening did not affect the

development of ethylene and respiration peaks, but delayed colour changes and

total volatile production. Further research into I-MCP application after

ethylene initiation is required to explain if the reduction in volatile production

would have any impact on consumers and industry.

Continued research would also allow studies to concentrate on extending

banana shelf life (stage 4-7)rather than the thoroughly researched banana green

life (stage 14). Extensions in the shelf life of banatras treated with I-MCP after

29

Chapter 2: Literature Rwiew

ethylene initiation have been fourd by Joyce et al. (1999), but additional

examination into the effects I-MCP has on banana eating quahty would benefit

the commercial market.

The use of 1-MCP as an ethylene antagonist is beneficial in prolonging banana

shelf life when applied early after ethylene application (Macnish et al., 1997 ;

Jiang et a\.,7999b;Joyce et al.,1999). However, the definition lvlzcnish et al.

(1997) and Jiang et al. (1999b) glve for their assessments of shelf life are from

green to eating ripe or using fruit softening, respectively, but these assessments

do not gauge the effect I -MCP has on consumer shelf life (stage 4 to 7).

Therefore a comprehensive study into the effect that I-MCP has on banana

colour (tureven degreening) and reduced volatile production with regards to the

commercial marketability of 1-MCP exposed fruit requires frirther investigation

2.8 Summary

As most bananas are harvested in an unripe state, the early detection of chilling

irUury would prevent unnecessary harvesting of unsaleable fruit. Early detection

could also allow for appropriate marketing and handling decisions to be made

preventing sub-standard fruit being sold to markets. Therefore, if either

ctrlorophyll fluorescence or PPO activity are determined to be signrficantly

different in chilled fruit compared to non chilled fruit, the findings would be

very useful to the banana industry.

Examining various temperature and ethylene regimes throughout the year may

reveal the need to use different ripening procedures when ripening bananas in

summer, compared to winter. This would allow the industry to optimise banana

quality and shelf life all year round.

30

Cløpta 2'. Literature Review

Fu¡thermore, posthalvest treatrnents applied to bananas such aS controlled and

modified atrnospheres, nihogen atnospheres, the application of l-MCP and

ethanol abnosphere storage after ripening have potential for extending product

shelf life. Extending the shelf life after ripening would greatly benefit the

industry as would delivering a better quality product to the consumer.

2.9 Research aims

The aim of this research project was to improve the postharvest quality and

extend the shelf life of bananas, by optimising the effect of temperatures and

ethylene levels in ripening rooms throughout the year. Quick and easy methods

for the early detection of chilling inju¡y among bananas were also investigated.

In addition, post riþening treahnents such as confolled and modified

atmosphere packaging, niüogen storage, the application of l-

methylcyclopropene at att early ripening stage and the storage of bananas in

ethanol ahospheres at later ripening stages were examined. These experiments

aimed to double the existing shelf life of bananas of from three to six days.

Banana quality was determined through a range of internal and external

measurements. Internal measurements included pulp firmness, soluble solid

concentrations and the determination of differences in banana aroma with the

use of a chemical nose. External measurements included visually assessing fruit

for chillinginjury, recordingthe general appearance of the peel andthe shelf life

(days from colour stage 4 to stage 7). The ultimate aim was to improve the

postharvest quality of bananas for industry and consumer satisfaction and to

continue the search for the 'perfect' banana, a banana that lasts longer all year

round.

31

Cheprer I

3. General Materials and Methods

3.1 Plant material

Cavendish bananas of the 'Williams' variety, were used for all of the

experiments in this project. Theywere grown using commercial growing

practices (8. Chartres, Chiquita Brands South Pacific, pers. comm., 1999) on a

single Tully property in North Queensland. The bananas were harvested at75Yo

maturity, at rþening stage I (hard, green appearance). They were harvested

from the middle of the banana bunch (hands 2 or 3) to ensure all were of a

wriform size and shape. Produce was then packed in cartons, layered with

plastic to protect the hands from rubbing against one another and transported

over 3 or 4 days in monitored refrigerated trucks above l3oC, to the Adelaide

Produce Markets at Pooraka before being delivered to the Plant Research Centre

at the University of Adelaide, V/aite Campus.

3.2 Preparation of fruit prior to experimentation

Upon arrival at the Plant Research Centre all banana fingers were separated

from their hands, deflowered and crowns were cleanly cut offusing a sterile

surgical blade. Bananas chosen were of a similar size and shape to reduce

variation ¿tmong fingers. Each banana was assessed prior to experimentation for

disease and defects (defected fruit >2cm ffea per firger, T. Johnson, Chiquita

5¿

Chapter 3: General Materials and Methods

Brands South Pacific, pers. comm., 2000). The bananas that did not adequately

meet Chiquita's established quality assessment criteria (T. Johnson, Chiquiø

Brands South Pacific, per. coÍìm., 2000) were discarded. Al1 of the acceptable

bananas were then dipped for 1 min. in an aqueous frrngicidal solution

containing 0.55 mL L I Sportak@ fungrcide with the active ingredient Prochloraz

(Hoechst Schering AgrEvo, Glen kis, Victoria) and allowed to air dry (Wills er

a1.,1990). Each of the fingers was then randomly allocated to treatnent groups.

3.3 Möilttoring ethylerle levels

Ethylene levels were monitored using a gas chromatograph (Varian 3400,

Varian Associates Inc., Mulgrave, Victoria) connected to a flame ionisation

detector (GC-FID). A stainless steel column (60 cm x3.175 rtrm i.d.) packed

with Porapak Q \r/ith 80/100 mesh was used. The oven, injector and detector

temperatures were set at 50"C, 135"C and 150oC, respectively. Gas flow rates

were 300 mL min.-l for compressed air, 40 mL min.-l for hydrogen and 50 mL

min.-l for nitrogen, the carrier gas (Ikajayklanget a1.,2000).

A 1.9 FL Ll ethylene gas mixture (Boc gas, p-standard, Adelaide) was used as

a gas standard. One mL of the ethylene standard was injected to calibrate the

GC-FID, sample injections were automatically linearly related to the standard

peaks and ethylene concentration detemrined. Measurements were taken in

duplicate before venting and immediately after ethylene gassing.

3.4 Monitoring carbon dioxide and oxygen in storage atmospheres

Carbon dioxide and oxygen amounts were analysed by taking 1 mL samples

from containers. Carbon dioxide was identified using gas chromatogaphy

(Varian 3300, Vmian Associates Inc., Mulgrave, Victoria) with a thermal

^tJ

Chapter 3: General Matenals and Møhods

conductivity detector (GC-TCD). The GC-TCD had a45.7 mm x 1.5 m stainless

steel column containing a molecular sieve (Tlpe 5A\40160 mesþ, with 5 mL

min.-r heliurn as the carrier gas. Temperatures were set at28"C for the column

and 90"C for the injector and detector ports. The GC was calibrated using a

standardmixture of 20.9Yoo2,0.03o/o COz and 79o/oN2@OC gas, B-standard,

Adelaide). Meastrements were taken in duplicate and performed immediately

after gassing and prior to opening of the contarner.

3.5 External quality assessment

3.5.L Green life

Surface colour of banana peels was evaluated daily. Classification of each

banana ripening stage was according to the CSIRO banana ripening chart (1972;

Figure 3.1). Green life of bananas was a subjective measurement which

assessed the number of days it took bananas at ripening stage 1 (green, hard) to

ripen to stage 4 (more yellow than green).

3.5.2 Shelflife

Shelf life of bananas was evaluated using the surface colour of banana peels. It

was measured by recording the number of days bananas at ripening stage 4

(more yelloq than green) took to reach ripening stage 7 (yellow, lightly flecked

with brown; CSIRO, 1972).

34


ã;.ti-

þ

I

3

t\

il.

\-

Figure 3.1: The 8 ripening colour stages (CSIRO, 1972)

,,

a

35


3.5.3 Discolouration index

A chart (Table 3.1) was used to score the peel appearance of bananas at

ripening stage 6 (fully yellow, CSIRO, 1972). The scores were weighted and

averagedinto a discolouration index @t), usingthe formula (3.1) from Schina

et al. (1999), as data for each üeatrnent was normally distibuted and fell within

a naffow range. The fomrula uses the number of bananas with no

discolouration (a), slight (b), mild (c), or severe discolouration (d) compared to

the total number of bananas in each ûeatment (n).

(axo) + (¿xl) + (cxz) +(dxt)Dscolouration index (DI) =

"..(3 1)

36


Table 3.1: Discolouration score chart.

0

Peel score Visualsymptom

Assessment

Healthyfruit

Slishttydiscoloured orchilled

Milddiscolourationor chillinginjury

Severe

discolourationor chilling injury

Appearance

Yellow, nodefects

Dullgrey/yellowcoloured fruit

Red/brownscarring alongvascular tissue

Red/darkbrown marksall overbanana

1

2

J

5t


3.5.4 Weight loss

Bananas were initially weighed prior to ripening as well as at ripening stage 6,

allowing total weigþl loss over the ripening and heatunent periods to be

assessed.

3.6 Internal quallty assess¡mehts

3.6.1 Putp flrnness

Pulp firmness w¿ts assessed at ripening stage 6 (full yellow), the preferred

eating stage by consumers (Anonymous, Your gUide to greater profits, undated),

and the final stage of ripening before signs of senescence (brown flecks) appear.

Each bdnana was tested using a twist tester Gtgo.e 3.2,Departrnent of

Agricultural Engineering, Massey Univefsity, NZ). The twist tester is claimed

to be easier and more reliable then a penetroSeter on soft fleshed fruit

(Studman and Yuwan a, 1992).

For the twist tester measurements, each banána were cut ttansversely. One half

of the banana was then pushed (exposed prllp end first) onto the fixed blade and

rotated in an anticlockwise direction until the pulp tissue failed. The arm

attached to the blade lifted against gravity and the highest angle prior to tissue

failtue was recorded. This procedure was replicated using the second half of the

banana. All measurements were then converted into a crushing strength value,

using the formula below (3.2). Soft fruit have a lower crushing strength than

hard fruit (Hopkirk et aI.,7996).

M sin?a'b

.(3.2)

38

Chapter 3: General ll4aterials and Methods

crushing süength (Pa)

M: mÐdmum moment produced by the arm when e : 90' 0{.m)

radius of the blade (m)

b: width of the blade (m)

0 : maximum arm angle before tissue failure occuned o

39

Chapter 3 : General lVfi.terials and Methods

Figure 3.2: The twist tester

40

Chapter 3: General Mat€rials and Methods

3.6.2 Soluble solid codtent

Ameasure of soluble solid concentration (SSC %) was used to measure total

sugar content of banana pulp. All of the banana halves used in the pulp

firmness tests (section 3.6.1), were used in these SSC tests. Firstly a banana half

was trimmed so that the twist tester damaged pulp was removed. Atransverse

slice of the banana was then cut and this pulp was squeezed through muslin

cloth onto the glass surface of a hand held refractometer (range 0-30% sugar

w/w, Bellingham and Stanley Ltd., England). The SSC (7o) measurement was

then recorded and temperáture calibrated. This process was replicated with a

slice of the second half of the banana and repeated for each of the fingers in the

treaünents. When all measurements were recorded the banana halves were

retumed to their treaûnent for use in the banana aroma analysis using a

chemical nose.

3.6.3 Aroma analysis

Banana ¿roma was evaluated using a Hewlett Packard Chemical Sensor (IIP

4440, Hewlett Packard Company, Ltd., 1998, DE, U. S.A.), commonly lnown as

a 'chemical nose'. This instrument consists of a headspace autosarnpler (lIP

7694),linked to a quadruple mass sensor and comprising its own data collection

and Piroutte analysis package (Infometrix Inc., Woodenville, WA U.S'A)

After removing the exposed banana tissue by slicing a transverse section down

the exposed end of the banana halt 10 - l5 g of pulp from the freshly cut end

was removed from each bananahaff (I2 banana halves per replicate - 120 -180

g of pulp each replicate) and placed into a Moulinex blender (Model D721Q50,

Robinson Industries Ltd., Auckland, NZ.). The pulp from one treatment

replicate was blended for six, I second pulses to simulate chewing. Five g of

4t

Chapter 3: General lrdaterials and Methods

banana homogenate was added to a20 mL headspace vial, this step was

repeated for 10 replicate vials. Heptadecane was used as an internal standard,

with 5 pL of heptadecane was added to each of the l0 vials prior to their clostre

with black butyl rubber seals in aluminium crimp caps. Vials were then

randomly allocated into the headspace autosampler and left to equilibrate for 5

min. The sample preparation procedure was then repeated for the remaiting2

treatment replicates. An extra headspace vial was prepared as a 'dummy' and

placed in the first position of the headspace autosampler afm as a trial run to

prevent the initial false reading that occurs immediately after start-up.

Headspace autosample configurations used for the bananas included; an oven

temperature of 42oC,a 3 mL sample loop temperature of 50'C, a transfer line

temperature of 60oC, a headspace cycle time of 3.5 min., a vial equilibration

time of 20 min., a pressurisation time of 0.3 mirt., a loop fiIl time of 0.15 min., a

loop equilibrium time of 0.02 min. and an injection time of 0.5 min. Helium

gas was set as the carrier gas at 13.1 kPa with a flow rate of 30 mL min.-r and

vial pressure was set at96.5 kPa. The headspace autosampler/mass

spectrometer interface temperature was set at 90'C and mass spectrometer

parameters were set to scan a mass/charge (mlz) range of 49-200. The low m/z

was chosen to avoid signals from water, oxygen and less discriminating ions

produced by volatile chemicals.

3.6.3.1 Chemícsl nose statisticøl analysís

The raw data collected from the headspace and mass spectrophotometer was

analysed using the multivariate data analysis package, Piruotte. Principal

Component Analysis (PCA) was used to detennine aroma similarities in the

collected data and organise it into a PCA model. The PCA model forrned from

42


the chemical nose data was nofmalised and cross validated to ensure the

principal components (or factors) accoufited for greater than 80% of the total

variation between banana sÍtmples (Wirthensohn et a|.,1999)-

Score plots from the PCA model mapped the original data samples onto

principal component (factor) axes specified by loadings. The loading plots

represent the relative contribution ofevery original variable (ion) to a factor

axis. A loading of one meant that the variable (ion) coincided with a principal

component. Fruit with similar volatile profiles were grouped together on plots,

but separated from fruit that had different volatile profiles (Wirthensohn ef a/.,

teee).

Aroma compounds were identified using the variables (ions) that were

associated with the extreme ends of the factors from loading plots and matching

those variables with aroma compounds from an establishedvolatile compound

library.

43

Cheptëü 4

4. ire-ribenine Detection of Chilline IniurY

4.1 Introduction

Physiological changes occur within bananas when they are subjected to

temperatures below 13"C (Olorunda et a\.,1978; Patterson, 1980; Paull, 1993;

Wills e/ at.,1998). Breakdown of tissue andthe disruption of metabolic

pathways caused by exposure to low (<13'C) temperatures affects banana taste,

flavour, appearance and their nutritive composition (Stover and Simmonds,

l9S7). This physiological disorder is commonlyknown as chilling tnjury'

A chilling irijury sSmptom that is visible to the naked eye is the browning of

vascular tissue (Maniott, 1980, Stover and Simmonds, 1987; Kays, 1991; Wang

and Gemma,1994). This syrnptom appears as either red or brown coloured

streaks dlong the peel of mild to sevetely affected fruit or as a dull yello#grey

colouring on the entire peel of slightly affected fruit. These obvious symptoms

reduce the commercial acceptability of bananas, because consumers relate

colow to the overall quality of produce 6ays, 1999). Consequently, early

detection of chilling rnjury would enable the market to redirect chilled fruit to

either markets that accept lower gade fruit or dispose of them accordingly.

44

Chapter 4: Pre-ripentng Detection of Chi[ing Injury

While many studies have been concerned with preventing chilling injury (Jones

et at.,1978) and understanding what occurs in fruit to cause chilling ittj.ny

(Wills et a1.,7975;Torasker andModi, 1984; Wang and Gemma, 1994)' few

studies have researched methods for the early detection of chilling injury'

The biochemical processes which lead to the browning or discolouration of

chilled fruit have been reported to be enzymatic oxidation by

polyphenoloúdases (PPOs) in the cytoplasm, after phenolic compounds like

dihydroxyphenylalanine (DOPA) leak out of the vacuole (Jones et a1.,1978;

Amiot et al.,1997). PPOs have also been found to be inüacellularly bound to

chloroplasts (Shomer et a1.,1979;Amiot et aI.,1997) and cell walls QVIarques ef

al. , 1995). Amiot et at . (1997) also reported that browning in fruit always

occurs after physical changes to plastidic and vacuolar membranes, allowing

enzqe and strbstrate to interact. This then leads to browning due to the

formation of dark brown pigments,lnown as melarrins @almer, 1963). A good

correlation between browning and higher PPO activity was found in inadiated

bananas (Thomas andNair, 1971; Amiotet a1.,1997). This srlggeststhatPPO

activity mdy be a usehrl method of detecting chilling injury which affects

conformational changes of cells within tissues that produce browning as a

result. However, datahave also been reportedby Gooding et al. (2001)that

show peel PPO activity is high during early development and then declines

when ripening begins, where it remains constant' They also found no

differences in PPO activity after induced wounding. As these studies found

conflicting results, fi¡ther study into banana PPO activity is required.

As membranes are disrupted by chilling injo.y, the chloroplasts may also be

disrupted resulting in changes to the photosynthetic ability of the tissue. This

disruption may be detected via chlorophyll fluorescence, which is a non-

45

Chapter 4: Preripening Detection of Chilling Injury

destructive, eflective method of measuring the primary processes of

photosynthesis that take place in the chloroplasts @eEll et al.,1999). In fact

there is some evidence to suggest chlorophyll fluorescence does detect chilling

injury in bananas, with Smillie et al. (1987) using aFp(maximal rate of increase

in induced chlorophyll fluorescence) measurement. Smillie et al. (1987)

indicated bananas held at 0"C had significantly decreased rates oflog Fp than

those at 13oC, but the severity of the injury induced at OoC may have aided in

the detection of the change in measurements. Milder cases of chilling injury

that commonly appear on fruit in indusfy have not been investigated using

chlorophyll fluorescence. Since this research smaller, easier and portable

fluorescence detectors have become accessible making industry use a possibility

by measuring various parameters (detailed in section 2.4.1.1) that have yet to be

examined in relation to detecting chilling injury in bananas.

Chilling injury is currently identified in industry by the visual qrnptoms

mentioned previously, but these signs often do not aþþear ptior to riþening

unless severe chilling (eg. 5 days at 5"C) occurs in the preclimacteric phase

(Stover and Simmonds, 1987). The aim of this studywas to use chlorophyll

fluorescence and PPO activity measurements as possible alternative methods for

the detection of chilling injury in bananas that could be used by indusûy prior to

the appearance of visual symptoms.


These two experiments were conducted lli'7999. All bananas were harvested

and prepared as described in sections 3.1 and 3 .2,but were not ethylene treated

in either of the following experiments.

46

Chapter 4: Prerþening Detection of Chilling Injury

4.2.1 Chlorophyll flüorescence

Chlorophyll fluorescence induction was measured on chilled and non-chilled

Cavendish bananas cv. William.s. Initially the bananas at ripening stage 1 (hard,

geeÐ were assessed using the Royal Horticultural Society ßHS) coloru charts.

Areas on the banana of the same colour were used for the chlorophyll

fluorescence measurements. Those areas \ilere dark adapted for 30 min. prior to

fluorescence induction, using a dark adaptor clip þreventing light from

accessing the surface area of the banana prior to fluorescence induction). After

dark adaptation the fluorescence measurement was taken with a Fluorescence

Induction Monitor (FIM 1500) (ADC. Bioscientific Ltd., Hoddesdon, England)

ata7\o/olight level. The dark adaption period of 30 min. andthe 70%lig!Í

level were found to be suffrcient in prelimiharytrials.

After initial fluorescence mea$rement, bananas were placed into unsealed

polyethylene bags and stored either at 5"C (chilled) ot at22"C (non-chilled).

Chilled bananas were stored at 5"C for 5 days, but removed daily for chlorophyll

fluorescence measurements. After the 5 day cool storage, chilled bananas were

stored at22'C (room temperature) until the end of the experiment.

Chlorophyll fluorescence measurements were done daily; with bananas stored

at 5oC brought out to 22"C to equilibrate to room temperature prior to

measurements ensuring accurate and comparable fluorescence measurements

with fruit held continually at 22"C. Visual assessment of banana ripening stages

as well as the chlorophyll fluorescence measurements, were recorded dailyuntil

all bananas reached ripening stage 4 (more yellow than green).

ARandomised Complete Block Design (RCBD) was established with six

hands representing six replicates. Each of the six hands were separated into

47

Chapter 4: Preripening Detection of Chi[ing Injury

halves and randomly allocated into either chilled or non-chilled treatrnent

groups. Treatrnent groups consisted of either two or three experimentål units

depending on the number of fingers makingup the hand.

Replicates were analysed for changes in each of the chlorophyll fluorescence

parameters @o, Fv, Fm, FvÆm, Tf max and area detailed in section 2.4.1.1), on

chilled and non-chilled bananas from ripening stage I to ripening stage 4. Each

replicate was analysed in a simple linear regression (with gfoups) to eliminate

the variability between the hands. Regression results were then analysed in a

one-way ANOVAwith replicates using a t-test. Results from ripening stage 1

were also analysed separatelyusing a general analysis of variance with

replication. The number of days it took for the chilled or non-chilled bananas to

reach ripening stage 2 was the covariate factor. A simple t-test was used for the

analysis with the Genstat 5.41 program (Release 41,4h ediion, 1998, Lawes

Agricultural Trust, Rothamsted Exþerimental Station).

4.2.2 P olyphenoloxidase actlvlty

Polyphenoloxidase (PPO) activrty was measured using a digltal oxygen

electrode system (Model 10, Rank Brothers Ltd., Cambridge, England).

Bananas at ripening stage 1 were assigned to either a chilled treatment and

stored at 5oc for 4 days (until visible symptoms of chilling injury appeared)

prior to PPO evaluation or to a control (time 0), non-chilled treatnent where

PPO activity was tested immediately. The PPO activity of each banana was

examined in duplicate.

Two pH 6.8 buffer solutions were required for the measurement of PPO. The

first 100 mL solution was an 'extract' buffer made up of l:4 v/v of 0.1 M citric

acid and 0.2 M di-sodium hydrogen orthophosphate dihydrate. The second pH

48

Chapter 4: Preripening Detection of Chilling Injury

6.8 buffer prepared was the 'run' buffer, it was made exactly the same way as

the extract buffer above, with the only difference being the addition of 1% (wþ

sodium dodecyl sulphate (SDS) to the solution.

Each reaction took place in the 'run' buffer at pH 6.8. The 'run' buffer (1950

¡rL) was initially added to the reaction cell of the digital oxygen electrode

system. The 'run' buffer was constantly stirred and warmed to 25"C by a

continuous flow of thermostatically controlled water.

Preparation of the peel sample excised from the neck of the bananaproceeded

by gnnding 0.3 g of the tissue into a paste using a mortar, pestle and liquid

nitrogen. To the paste, I mL of extract buffer was mixed and a further 2 mL of

extract buffer added to the mixture until a fine slurry was formed. A 1 mL

quantity of the sample mixture was then added to the pre-warmed reaction cell

and the oxygen electrode stabilised at95o/o 02 content.

The substrate used for this PPO analysis was 100 mM 4-methyl catechol.

Oxygen consurnption by PPO in the banana sample was initiated when 50 pL of

100 mM 4-methyl catechol was added to the stabilised reaction cell. The

oxygen cell ouþut (mV) was then plotted onto a chart recorder (Omni Scribe,

Austin, Texas). PPO reaction rates were calculated into nmoles min.-l g.f.ú.-l.

The reaction cell (3 mL) held 774 nmoles of dissolved oxygen and the resulting

plot was dependent on the PPO activity in the banana sample (g.f.wt.). The rate

of the reaction (nmoles oxygen consumed mitt.-t) was determined using the

initial slope of the resulting plot (formul a 4 .l). After each reaction the cell was

cleaned with deionised water and the entire procedure repeated.

49

Chapter 4: Preripening Detection of Chi[tng Injuy

PPO activity (nmoles min.-l g.f.!vt-t)

:774 nmoles oxygen / rate of reaction I g f.wt (4 l)

The experiment was aRCBD with six replicates. One hand comprised one

replicate. Each hand was randomly divided into chilled and non chilled

treatrnent groups similm to the chlorophyll fluorescence design. Measurements

were onlyperformed on peel from the neck of the banana as visual s5rmptoms of

browning (PPO activrty) could be clearly seen. The PPO measurement for each

finger was performed twice. PPO reaction rates from each of the measurements

were analysed using a t-test with the Genstat 5.41 program (Release 4.1 , 4ú

edition, 1998, Lawes Agricultural Trust, Rothamsted Experimental StatioÐ.

4.3 Results

4.3.1 Chtorophyll fluorescence

The heated bananas held at 5oC for a period of 5 days displayed chilling

injury symptoms of brown vascular streaking on the peel of the bananas. All

chilling treated fruit after 5 days were affected with red or brown vascular

streaking. Fruit held at room temperature (22"C) did not have any visible

browning s5rmptoms of chilling injury $igure 4.1). Similar chlorophyll

fluorescence measurements for chilled ætd non-chilled fruit were recorded

throughout ripening stage 1 (Table 4.1).

50

Chapter 4: Pre-rþening Detection of Chi[ing kiury

Figure 4.1: Peel appeafance of Cavendish bananas cv. Williams after a holding

period of 5 days at (Ð 5"C afid (B) 22'C.

Table 4.1: Chlotophyll fluorescence rheasurements on Cavendish bananas cv

Wliams recorded daily duting chilling exposure treafnent at 5oC for 5 days

and ripenin g at 22'C at ripening stage 1.

Average chlorophyll fluorescence measurement at

ripening stage l"

Chlorophyll fl uorescence

parameter

Chilled bananas

(5"C for 5 days)

Non-chilled bananas

Q2"C)

Fo

Fm

Fv

Fv/Fm

Tf max (ms)

Area

382 a

1799 a

l4l7 a

0.785 a

718 a

21,035 a

368 a

162l a

1253 a

0167 a

929 a

23,866 a

"Means are of 6 replicates; the same subscript \ rithin rows denotes similar

chlorophyll fluorescent measurements at P>0.05.

51

Chapter 4: Preriperiing Detection of Chdlmg Injury

All fluorescence parameters declined \Mith time as the fruit lost chlorophyll

during ripening from stage I to 4. However, the rates of decline of the

chlorophyll fluorescence pmameters did not vary in response to chilling injury

of bananas (Table 4.2).

Table 4.2zThe daily change in chlorophyll fluorescence parameters measured

on Cavendish bananas cv. Williams that were stored at 5oC for 5 days followed

by storage at22"C until ripening stage 4, or stored at room temperature only

(22"C) until ripening stage 4 (more yellowthan greÐ.

Change in parameter per daf

Chlorophyll fl uorescence

paræneter

Chilledbananas

(5"C for 5 days)

Non*hilledbananas

(22"C only)

Fo

Fm

Fv

FvÆm

Tf max (ms)

Area

-39 a

-481 a

431a

-0.172 a

-1.42 a

4,864 à

-18 a

-437 a

-437 a

-0.147 a

-1.42 a

4,864 a

'Means are of 6 replicates; the same superscript within rows denotes similar

changes in parameters at P >0.05.

52

Chapter 4: Preripening Detection of Chillmg Injury

4.3.2 P olyphenoloxidase

Polyphenoloxidase (PPO) activrty was measured in banana peel of chilled and

non-chilled bananas. Hoyever, the activity of PPO in chilled fruit was not

different to that of non-chilled fruit (Table 4.3).

Table 4.3: The pollphenoloxidase activity in the vascular tissue from the peel

of Cavendish bananas cv. Williams stored at ripening stagel at 5oC for 4 days,

compared to bananas stored at22"C for 1 day at ripening stagel.

Storage feátment Polypherioloxidase rate atripening stage I'

(nmoles min.-l g.f.wt-t)

Chilled bananas

Non chilled bananas

263 a

252 a

'Mearts are of 6 replicates; the same subscript denotes sittrilar

polyphenoloxidase activity at P>0.05.

53

Chapter 4: Preripening Detection of Chi[ing Injury

4.4 Discussion

Chlorophyll fluorescence measurements reported in this chapter could not be

used to detect chilling i"j".V in Cavendish bananas cv. William,s, even where

visual symptoms of the disorder were obvious. The localisation of the browning

pigments associated with chilling injury was in the sub-epidermal vascular

tissue, whereas chlorophyll fluorescence measurements were taken on

epidermal+hlorophyll containing tissues. It seems likely that the photosynthetic

chloroplasts which are located in epidennal cells are less chilling sensitive than

the vascular tissues where the enz¡mes and substrates required for browning are

localised. Therefore enzymatic browning is unlikely to be associated with

photosynthetic changes, and so chlorophyll fluorescence measurements are not

a good indication of mild chilling in compárison to severe chilling where

chloroplast disruption can be measured by fluorescence (Smillie et al., 1987).

Chloroplast function deteriorated in cells due to severe chilling injury, leading

to decreases in chlorophyll fluorescence emissions (Smillie and Hetherington,

1990). A change in chlorophyll fluoiescence due to ripetting rather than chilling

injury was observed; this may have been due to the parameters measured

(Smillie and Hetherington, 1990). The chlorophyll fluorescence instrument

available for this experiment determined which parameters could be measured.

Fp that was determined to indicate severe chilling inj".V (Smillie et aL.,1987)

could not be measured. However, results in section 4.3.1 indicate that F6, Fy,

F¡a, FyÆ¡a, Tf*u* and Area parameters were unsuitable chilling itjory indicators

for mafine green bananas.

PPOs are bound to chloroplasts and cell walls (Shomer et al., 1979 Marques e/

al. , 1995;Amiot et al., 1997), but browning reactions ensue when the enzqe

54

Chapter 4: Prerþening Detection of Ct"tlglryty

becomes mobile and able to interact with substrates from plaSids and vacuoles

due to chilling rn:ury and subsequent membrane leakage (Stover and

Simmonds, 1987; Wills et al., 1998). The resulting brown substances are

distributed around the vascular tissues (sieve tubes) of banana peels (Mwata,

1969). Similarly chilling injury in tomatoes was also observed around the

vascular tissue of fruit (Hong and Gross, 2000). Therefore, it seems likely that

the sieve tubes that are present in the phloem Íre sensitive to chilling stress.

The sensitivity of sieve tubes to chilling shess maybe due to their cell anatomy

consisting only of cytoplasm without a nucleus (Salisbury andRoss, 1985) and

therefore little ability to carryout cellular repairs after chilling stress has

occurred.

The visible build-up of brown pigments in the sieve tubes around the vascular

tissues of the fruit increase in ntrmber with chilting severity (Murata, 1969)

gving rise to the various degrees of chilling injury symptoms observed on

banana peels. A low build-up causes slight discolouration with a higher build-

up causing severe discolouration, although the peel in severely chilled fruit is

generally discoloured as well as the vascular tissue. Apossible method

investigated to detect the early appearance of this discolourdtion was to measure

the PPO enzyme activity in the peel of chilled fruit.

PPO activities in chilled as well as non-chilled mature green bananas at stage 1

were similar, contary to studies by Thomas and Nair (1971) that reported a

correlation between browning and the activity of PPO in bananas upon injury

with gamma irradiation. This was thought to be due to chilling injury causing

cellular leakage after cell membrane degradation, allowing the PPO enzyme to

mix freelywith its subsfiates, rather than increasingPPO activity.

55

Chapter 4: Preripemng Detection of Chi[ing Injury

Changes in PPO activity in bananas \À/ero reported by Ornuaru and Izonfuo

(1990), where ripe peel had an 18-fold increase in PPO activity, compared to

green peel. This was thought to be due to cellular leakage after cell membrane

degradation, allowing the PPo en'4lme to mix freely with its subsfrates'

However, Amiot et al. (1997) and Goodinget al. (2001) reportedthatPPO

activity does not increæe during storage, so Omuaru et al. (1990) may have

confirsed PPO activity with an increase in the browning reaction due to substrate

leakage during ripening. Gooding et al. (200I) reported that banana PPO

activity retnained constant at all ripening stages, arrd after 72hrs after inducing

mechanical wounding only a slight increase irt banana peel PPO activity and no

increase in flesh PÞO activity was found. They concluded that as the peel

increase was negligible in wounded fruit when compared to non-wounded fruit

the wound defence response of increased PPO activity is not present in bananas.

This possibly explains why PPO activity did not increase in chilled fruit as.:

reported in sectioh 4.3.2.

It is also posslble that the PÞO activity o!'tron-chilled' fruit used in this

experiment had béen increased in the field by being exposed to in-field chilling

temperatures. Bananas were háfVested in JUly after a month where the daily

temperature dropped below the cfitical chilling temperature of 13"C on two

consecutive days @ureau of Nrleteorology, 2000-01). This mayhave already

increased the PPO activity in the peel similarly to that of the postharvest chill-

induced fruit. Howevet, as both þeet anO flesh contain high levels of PPO

isorymes throughout their deveibpthent (Goodifi g et a1.,2001); it is still unlikely

that this technique would be usefril for eady detection of chilling injury.

56

Cløpter 4: Pre-rþening Detection of Chilling Injury

4.5 Conclusion

Meastring chlorophyll fluorescence levels or PPO activity didnot allow

discrimination between chilled or non-chilled bananas. Mild chilling injury is

of most concem to wholesalers and markets, but unlike severe chilling injury,

the chloroplast disruption caused by mild chilling is not measurable using

fluorescence. Measuring PPO activity was also found to be an unsuitable

method of chilling injury detection, as PPO activity is alreadyhigh and is little

affected by physical and physiological stresses. Further research into the early

detection of chilling injury is required to ensure Australian winter grown

produce most susceptible to chilling injury are handled appropriately. This may

mean lowering prices, discarding produce (if better produce is around) or

ripening fruit at higher temperatures (see chapter 5).

57

Chapter I

5.1 Introduction

Ripening of bananas in commercial practice is regulated by adjusting npening

temperature, ethylene concentration and humidity to achieve a zuitable ripeness

within a planned period of time to satisfu retailer demand. Suggested banana

ripening temperahres differ, including I 4-21' C (Palmer, I97 I), 16-17 " C

(Maniott, I 980), 14-24" C @eacock, I 980), I 8-20"C (Salunkhe and Desai,

1984), 16-21'C (Israeli andLahav, 1986) and 16-18'C (Turner, 1997). These

are all adequate ripenitrg temperatufes for Cavendish bananas (cv. Williams),

but maynot be optimal in all seasons of the year.

Palmer (I97I) suggests th¿t there is considerable diffrcuþ when predicting the

ripening behaviour of h¿rvested baflanas in Australia due to large temperature

variations between seasons. The e$ect of summer and wirtter harvesting on

optimun ripening temperatures of bananas in Ausüalia (QLD and NSW) was

briefly investigated by Young and colleagues between 1929-31 (Yowtg et al.,

1932),and later by Rippon and Trochoulias (1976). Young et al' (1932)

recommended ripening temperatures of 20 or 2l"C in sunmer and l9'C for

58

Chapter 5: Optimising Ripemng Temperatures and Ethylene Concentrations Tlroughout the Year

winter. Rippon and Trochoulias (1976) agreed that in Australia l9"C was best

used for winter bananas, but found that harvested summer bananas should be

ripened at 17"C. However, only two quality parameters (shelf life and pulp

firmness) were measured @ippon and Trochoulias,l9T6). Other important

commercial parameters, such as visual peel appearance and eating quality,

should also be considered (Israeli and Lahav, 1986).

Bananaripening is sensitive to exogenous ethylene gas @eacock,l972),:urrilr

levels of as little as 0.1 pL Lr for Z4hrsable to induce ripening (W1l7s et al.,

l99S). However, commercial npening enterprises use the higher rate of 300 ¡rL

L 1 of ethylene to ensure a uniform and rapid rate of ripening initiation

@.Charhes, Chiquita Brands South Pacific, pers. comm .,1999). There is no

evidence, however, of the effect various levels of ethylene may have on the

quality of bananas ripened throughout the year

The aim of this study was to reþort the quality effects of various ripenltrg

temperatures and exogenously applied ethylene levels on fruit harvested in

different months of the yeaf. Extefnal dnd internal quality parameters including

banana green life, shelf life, peel appearance, pulp firmness, soluble solids and

aroma were measured to détermine optimal banana quallty results from industry

ripening procedures.

5.2 Materials and méthods

5.2.1 Plant material and procedure

Cavendish bananas cv. Williams were harvested in altemate months of the year

from January to November 2000. Fruit from one property in Tully, North

Queensland, were hansported and prepared as detailed in sectíons 3.1 and3.2.

59

Chapter 5: OptimisirE Ripening Temperatures and Ethylene Concentratiors Throughout the Year

After fingerpreparation, bananas were randomly allocated to ripening

treatrnents.

Treatnents were applied in sealed 10-L plastic containers that cont¿ined six

banana fingers, a carbon dioxide scavenger (160 g ofCa(OII)2) and a saturated

KNO3 solution to maintain a relative humidity of 95o/o. Containers were

randomly allocated to monitored, temperature confrolled rooms.

5.2.2 Etrect of ripening temperature and harvest months

Similar to commercial practice @. Chartres, Chiquita Brands South Pacific,

pers. comm.,lggg),bananas were ripenedatl4,16, 18 or20"C after ethylene

gas injections produced a calculated concentration of 300 ttl- Ut ethylene in

each container. After 24hrs,the containers \ilere vented for 30 min., and

ethylene was reapplied. Thereafter, containers were vented daily, without

ethylene application, until fruit were more yellow than green Gtage 4). Bananas

were then placed into vented poþthylene bags at22"C and assessed daily until

fruit staned to senesce, showirtg a yellow peel colour with light brown flecks

(stage 7).

5.2.3 Effect of ethylbde concedtþations and h¿fvest months

Bananas were ripened at l6'C with 50, 300 or 1000 pL L1 ethylene gas. After

24hrsthe containers were vented for 30 min. and ethylene was reapplied.

Ethylene levels applied were monitored as described in section 3.3, using a gas

chromatograph connected to a flame ionisation detector. Containers were

vented daily after 48 hrs of ethylene gas, without frirther ethylene reapplication.

At ripening stage 4 (more yellow than green) batranas were removed from their

containers and placed into polyethylene bags at22'C and assessed daily until

60

Chapter 5: Optimising Ripening Temperatures and Ethylene Concentrations Tlnoughout the Year

they reached ripening *age 7 (full yellow with light brown flecks)

5.2.4 Quality assessilents

Treated bananas were assessed for green life, shelf life, peel discolouration,

total weight loss, pulp firmness, soluble solid concentrations and aroma analysis

as outlined in the general materials andmethods (sections 3.5 and 3.6).

5.2.5 Statistical assbssiledt

Every 2 months of the year 20001 2 simultaneousþ run experiments were set up

with bananas placed atthe 4 temperatures or 3 ethylene concentrations, with 3

different plantation blocks froln I property acting as replicated, randomised

complete blocks. Every ripenirrg temperature and ethylene concentration

treatnent for each reþli,cate contditred l2bananafngers; 6 were used for

extemal assessments ând the remâining 6 were used for intemal assessrnents.

Data, except aroma dáta, were analysed with the Genstat 5 program @elease

4.!,4ú edition, 1998, Lawes Agricultural Trust, Rothamsted Experimental

Station) using the Gener¿l ANOVA directive for two factors, harvest date and

ripening temperature or haivest ddte and ethylene concenfiation. A least

significant difference test (LSb) dtthe 5o/olevel was used to determine

signif,rcant differences between means.

APirouette analysis package (Infometrix Inc, Woodenville, WA, USA) using a

Principal Component Analysis (PCA) was used to detennine any ¿ìroma

differences as describedin section 3.6.3.1.

6l

Chapter 5: Optimising Ripening Temperatures and Ethylene Concentrations Tlroughout the Year

5.3 Results

5.3.1 Climatic data for Tully, North Queensland

Dailyminimum temperatures were recorded at the South Johnstone research

station for the month prior to harvest; the graph is missing some data as not all

dailymeasurements were recorded @gure 5.1, Bureau of Meteorology, 2000-

2001). The months before the July and September harvests show 4 and 9 days,

respectively, on which the daily minimurn temperature dropped to l3oC or

below. All other minimum field temperatures recorded in the months prior to

harvest were between 15 and 25"C.

Two cyclones crossed the northern Queensland coast betwe en25-29hEebruary

and l-3'd April 2000 (cyclones indicated on Figure 5.2-5.4). Cyclone Steve, a

category 2 cyclone, hit the coast on the25h February, with wind gusts of 125-

170 lan hr 1, causing considerable structural darnage to banana plants used in

this research project (T. Johnson, Chiquita Brands South Pacific, pers. comm.,

2000). The second cyclone, Cyclone Tessi, was a category I cyclone with

winds that did not reach above 125 kn hr I resulting in minimal sfuctural

damage.

62

Chapter 5: Optimising Ripening Temperatu¡es and Ethylene Concentrations Throughout the Year

-+January 20OO

-+ May 2000 4l-July 2000{-November20002000

nt 13'Cti

30

25

6,20oãa!

8-EI 'tsEaEE.E

àrooo

5

31 29 27 25 23 21 19 17 15 13 11

Days þliot to harvest lnonth

97531

Figure 5.1: The daily minimurh air tdrhpe¡afures ("C) from South Johnstone

Research Station @ureau of Meteorology, 2000-01) for the month prior to

harvest. Minimum daily temperatures were not recorded on days missing

appropriate synbols.

q

63

Chapter 5: Optimising Ripening Temperatures and Ethylene Concentrations Thrroughout the Year

5.3.2 Effect of ripening temperature and harvest months

Increased green lives (ripening stage I - 4) were observed for lower riperung

temperatures throughout the year (Figure 5.2), with bananas ripened atl4"C

requiring 30{,0% longer to ripen to stage 4 than bananas ripened at 16, l8 or

20oC. Green lives of bananas ripened at 18 and 20oC were similar within

months, but were l-2 days longer in September and November compared to

other harvest months. Similarly bananas ripened at 14 and l6oC, had slightly

longer green lives in September andNovember compared to those ripened in

January, March, May and July. The maximum green life of ll days was

achieved in September using a ripening temperature of l4oC, and the minimum

green life of 3 days was achieved in January using a ripening temperature of 18

or 20oC.

&

Chapter 5: Optimlsing Ripening Ternperatures and Ethylene Concentrations Tluoughout the Year

Autumn \üinter Spring

Cyclone I Cydone 2

Summer

14

12

10

8

6

oot,oEtrd,oo

4

2

0

January March May July

Month hdrvested

September November

Figure 5.2: Green life (ripening stage 14) of Cavendish bananas cv. Williams

harvested bimonthly and ripened at 14, 16,18 or 20"C with 300 pLLl ethylene.

Each column represents the mean of 3 replicates consisting of 6 bananas each;

the bar denotes least significant differences between temperatures vvithin

months e<0.05). Climatic events are shown above.

a14"C I16"C tr18"C n20'CJ rsd (0.e)

65

Chapter 5: Optimlsing Ripening Temperatr¡res and Ethylene Concentrations Tlroughout the Year

Bananas ripened at higher temperatures (18 and 20'C) in January, May and

November hada16-370lo shorter shelf life than those ripened at lower

temperatures (14 and 16"C) (Frgure 5.3). In March and July, ripening atl4"C

resulted n a20-50%o increase in shelf life, compared to those ripened at 16, l8

or 20oC. Bananas ripened at the various temperatures during September had

similar shelf lives of about 6 days The maximum shelf life of 9 days was

achieved in May using a ripening temperature of 14oC, and the minimum shelf

life of 4 days was achieved in March using a ripening temperature of 20C.

66

Chapter 5: Optimísing Ripening Temperatures and Ethylene Concentrations Tkoughout the Year

Summer Autumn Winter Spring

12Cyclone 1 Cydone 2

10

I

4

0

January fllarch liay July

Month harvested

Septenùer Àlovenöer

Figure 5.3: Shelf life (ripening stage 4-7) of Cavendish bananas cv. Williams

hmvested bimonthly and ripened at 14, 16,18 or 20"C \ rith 300 t I Ut ethylene

Each column represents the mean of 3 replicates consisting of 6 bananas each;

the bar denotes least significant differences between temperatures within

months (P<0.05). Climatic events are shown above.

6o(!tto

=oa

2

t14"C r 16'C tr 18"C a2O'CJ .o ro.nl

67

Chapter 5: Optimising Rip€ning Temperatures and Ethylene Concentrations Throughout the Year

Fruit in January, May and November had a healthy, bright yellow peel

appearance indicated by the low (0 - 0.5) discolotuation index scores (Figure

5.4); these readings were similar for all ripening temperatures used. Ripening

temperatures in March, July and September affected banana peel appearance

(Figure 5.4), with ripening temperatures of 14 or l6oC resulting in more

discolouation than 18 or 20"C. Fruit harvested in March and July appeared a

geylsh yellow colour compared to those harvested in January, May and

November, while fruit ripened in September had a similar grey/yellow peel as

well as distinct red,/brown vascular streaking.

68



Cyclone I Cyclone 2

3

2xott.E

.9a!

-9ottoi5

0

January $larch Itihy Juty

Month harvested

Septenber ltvenöer


bimonthly and ripened at 14,16,18 or 20"C with 300 pLLl ethylene where, (0)

represents healthy or yellow peel, (l) slight yellow grey peel, (2) red/brown

vascular sfreaking along banana neck or (3) red/brown vascular streaking along

length of peel. Each column represents the mean of 3 replicates consisting of 6

bananas each, the bar denotes least significant differences between temperatures

withinmonths (P<0.05). Climatic events are shown above.

J14"C ¡ 16'C

tr18"C a2O"C

lsd (0.6)I

69


Bananas ripened at2}'Cwere 5-15%0 firmer than those ripened at l4"C nJanuary, March and May, but had similar f,rmness readings in July, September

and November (Figure 5.5). Average finmess readings were similm between

most months.

120

0


Month harvested

September November

Figure 5.5: Pulp fimrness of Cavendish banana pulp cv. [lilliams at ripening

stage 6, harvested bimonthly and ripened at 14,16,18 or 20'C with 300 pLLr


bananas each; the bar denotes least significant differences between temperatures

within harvest months (P<0.0 5).

140

00

6'È580.Dø(,trEÈ60_o.)À

40

20

a14"C I16"C tr18"C 120"C= lsd 15.4)

70


Overall, SSC varied between ripening temperatures, with bananas ripened at 14

or 16oC having a higher average of 23.0- 23.4% than those ripened at 18 or 20"C

with an average of 22.0Yo. Monthlytends showed an increase in SSC levels

dnringJulyand September (24%)with lower SSC in othermonths (21.0-

22.s%).

10

35

30

25

¡20õU'u, 15

5

0


Month harvested

September November

Figure 5.6: Soluble solid concentration of Cavendish banana pulp cv. Williams

at ripening stage 6, harvested bimonthly and ripened aI14,16,l8 or 20'C with

300 FL Ll ethylene. Each coltunn represents the mean of 3 replicates consisting

of 6 bananas each; the bar denotes least significant differences between

temperatures within harvest months (P<0.05).

a14'C r 16"C tr 18"C tr20"C1. lsd (0.9)

7t

Chapter 5: Optimisrng Ripenine Temperatures and Ethylene Concentrations Throughout the Year

Weight loss of bananas after ripening at various temperatures throughout the

ye¿ìr was significant with increased weight loss of 2.1% when ripened at the low

temperature of 14oC, comparedto 1.6%o atl6-20"C. This weight loss

difference was statistically signifrcant but not visually obvious.

Aroma of bananas ripened at 14, 16, I 8 and 20 oC were analysed by mass

spectroscopy at ripening stage 6. The PCA model used to compare these data

between ripening temperatures within and between months explained 92Yo of

the total variance with two factors, where 79%o and l3%o related to Factor I and

2, respectively. The majority of the vmiation between treatments was identified

by Factor I and so the results will focus on Factor I (refer to section 3.6.3.1).

All bananas ripened in July and September as well as bananas ripened at 14 and

16'C in November and those ripened atl|'C in March and May, were located

on the negative side of Factor I and correlated to ion 55 @gure 5.7 and 5.8).

All January ripened bananas, as well as those ripened at 16, 18 and 20"C during

March and May, and bananas ripened at l8 and 20"C in November, were

located on the positive side of Factor I and correlated with ion 61. Aroma

compounds were identified with the aid of an established volatile compound

libraryused in conjunction with the chemical nose loadingplot @gure 5.8).

Volatiles identified included, l-butanol, 3-methylbutyl ester, 2-hexenal, butyl

esters arìd ethyl acetate.

72

Chapter 5: Optimising Ripening Temperatures and Ethylene Concentrations Throughout the Year

4

n

t&

0

-n Ripe andunripe aroma Ripe and over-ripe aroma

0

w

Figure 5.7: PCA chemical nose score plot of Cavendish bananas cv. [4/illiams

September (S) or November (N). F,ach data point represents means of banana

samples for a single replicate. Ripening treatments consisted of 3 replicates.

2

n-n

1€M

1til/

14ûû(

73

ñtooõr

Chapter 5: Optimising Ripenirig Temperatures and Ethylene Concentmtions Tluoughout the Year

Factorl

Figure 5.8: PCA chemical noss loading plot of ions (variables) derived from

Cavendish bananas cv. Williams ripened at14,16,18 or 20'C in January,

March, May, July, September orNovember.

74


5.3.3 Effect of ethylene concentrations and harvest months

Ethylene concentrations did not affect green life, shelf life or discolouration

indexes, but quality results were variable throughout the year (Flgure 5.9, 5 . l0

and 5.11). Similarly to previous harvest month results September harvests had

significantþ higher discolouration indexes and green lives, but lower shelf lives,

compared to bananas harvested in other months of the year. Banana weight loss

varied between ethylene concentration and harvest months, with higher weight

losses found when fruit were ripened with the lower 50 pL L 1 ethylene

concentration averaging l.9o/o, compared to 1.7%o and I.60/o weight loss when

ripened \A¡ith 300 and 1000 lt[- L*\ ,reqpectively. Total weight loss from ripening

stage 1 to 6 averaged between 1.5 -2.1% of total banana weight, but it was not

visually obvious.

75


Summer Autumn \üinter Spring

Cyclone 1 Cydone 2

E9

lsd (0.3) El50 pUL I300 pL/L 11000 ¡tUL


Month harvested

September November

Figure 5.9: Green life (rþening stage 14) of Cavendish bananas cv. [üilliams at

ripening stage 6, harvested bimonthly and ripened at l6"C with 50, 300, or 1000

pL L-t ethylene. Each colurnn represents the mean of 3 replicates consisting of 6

bananas each; the bar denotes similarities between ethylene concenfrations

within months (P<0.05). Climatic events are shown above.

10

II7ø'

lú^tt

ÊsEE4O3

2

1

0

76



Cyclone I Cvdone 2

12

10

Eil Fil E

I lsd(1.0) 850 ¡.rL/L I300 pL/L t 1000 pL/L


Month harvested

September November

Figure 5. 10 : Shelf life (ripening stage 4 -7) of Cavendish bananas cv. Williams

at ripening stage 6, harvested bimonthly and ripened at 16"C with 50, 300, or

1000 pL Ll ethylene. Each column represents the mean of 3 replicates


concentrations within months (P<0.05). Climatic events are sho\iln above.

8

6

ø,

(!ttú,!=

õat) 4

2

0

77

Chapter 5: Optimising Ripening Temperature and Ethylene Concentrations Throughout the Year

Summer Aufumn Winter Spring

Cyclone 1 Cyclone 2

3

2

xott.EEoo

=oõooo t il rlI

H tI

tr50 pL/L 1300 UL/L I1000 pUL

lsd (0.7)

0


Month harvested

September November

Figure 5.11: Discolouration index of Cavendísh bananascv. Williarms harvested

bimonthly and ripened at l6"Cwith 50, 300 or 1000 pL L 1 ethylene where, (0)

represents healthy or yellow peel, (1) slight yellow grey peel, (2) redibrorvn

vascular steaking along banana neck or (3) red/brown v¿rscular streaking along

length of peel. Each column represents the mean of 3 replicates consisting of 6

bananas each; the bar denotes similarities between ethylene concenfrations

withinmonths (P<0.05). Climatic events are shown above.

78

Chapter 5: Optimisrng Ripening Temperatures and Ethylene Concentrations Tlroughout the Year

Similarly, average SSC were similar for bananas ripened with 50 and 300 pL LI of 23%o compared to 22o/owhen ripened with 1000 pL Ll . However, bananas

from July and September harvests averaged higher SSC of 24%, compared to

those harvested in January, March, May and November of 22-23Yo (Figo.e

5.13).


Month harvested

September November

Figure 5.13: Soluble solid concenfations of Cavendish banana pulp cv.

Williams at ripening stage 6, harvested bimonthly and ripened at l6'C with 50,

300, or 1000 pL Lr ethylene. Each colunn represents the mean of 3 replicates

consisting of 6 bananas each; the bar denotes least significant differences

between ethylene concenfrations \ /ithin months (P<0.05).

35

30

25

Szo8rcal,

10

5

0

r 1000r300UL850

80


Banana ¿ìromas after ripening with 50, 300 and 1000 pLLl ethylene gas at

16oC were analysed when fully ripe using a chemical nose. A PCA model was

used to compare data of bananas ripened with the various ethylene

concentrations within and between harvest months, with the resulting plot

(Figure 5.14) explainng92% of the total variance with 2 factors. Factor I

related to 84Yo, while Factor 2 relatedto 8o/o of the variance. Again Factorl

comprised the majority of the variation, so it will be the focus of the results

(refer to section 3.6.3.1).

Ethylene concentrations did not affect banana aroma, as all ethylene treated

fruit from the same harvest month clustered in groups on the score plot

(Figure 5.I4), indicating similar profiles. However, similarly to Figure 5.7

and 5.8, July, September and November ripened fruit aromas clustered on

the negative side of Factor I correlating to ion 55, while January, March and

May harvested fruit aromas clustered on the positive side of Factor 1 relating

to ion 61. The loading plot for ethylene concentrations was the same as

observed in Figure 5.8. Aroma compounds identified in the ripening

temperature experiments including, 1 -butanol, 3 -methylbutyl ester, 2-

hexenal, butyl esters and ethyl acetate were similarly identified in this data

analysis.

8l

NoûL


-20 0

Factorl

20

Figure 5.14: PCA chemical nose score plot of Cavendish bananas cv. Williams

ripened aI"l6'C\4'ith 50 (5), 300 (3) or 1000 (1) pL Lr ethylene gas in January

(J), March (M), May (MY), July (JU), September (S) or November (N). Each

datapoint represents means of banana sarnples for a single replicate. Ripening

treatments consisted of 3 replicates.

1MY

tì,1Ysr'fiM

82

Chapter 5: Optimising Ripening Temperatures and Ethylene Concentrations Ttroughout the Year

5.4 Discûssion

Green life and shelf life are important commercial pa.r¿rmeters, but visual

appearance is equally as important. Even though ripening temperatures of 14

and 16'C extended shelf life in most months, the resulting fruit appearance was

not always appealing. Low temperatures slow down respiration and metabolic

rates which ultimately extends the entire ripening and senescence process

(Couey, 1982; Wills et a1.,1998).

Rippon and Trochoulias (1976) reported ripening temperatures were critical in

determining shelf life of bananas harvested throughout the year from NSW,

Australia. Holding temperatures after ripening had little influence on shelf life,

although holding temperatures of 10'C or higher than the initial ripening

temperatures prevented fruit from ripeningnonnally. Contrary to the previous

observations, fruit harvested in September had similar shelf lives when ripened

at74-20"C. Fruit harvested in September grew and developed during winter

and were affected by field chilling @tueau of Meteorology,2000-2001), which

has been found to directly or indirectlyreduce the shelf life of bananas (Seberry

and Harris, 1993). This would account for the similar shelf lives in September

when ripenedatl4-20"C compared with other months, when ripeningat14 and

16"C extendedbanana shelf life byup to 50%.

Chilling injury is a physiological disorder of bananas caused by temperatures

below l3"C for cv. Williams that lowers their visual appearance due to, for

example, subepidermal browning upon fruit ripening (Olorunda et a1.,1978;

Israeli and Lahav, 1986; Stover and Simmonds, 1987; Wills er a1.,1998).

Subepidemral browning is caused by enzymatic oxidation of dopamine

generally by polyphenoloxidase (Murata,1969). This accowrts for the high

83

Chapter 5: Optimising Ripening Temperatures and Ethylene Concentrations ThLroughout the Year

levels of peel discolouration observed in July and September harvested fruit that

grew and developed during winter. Field temperatures in the month before

these harvests dropped to near or below l3"C on 4 and9 days, respectively

(Bureau of Meteorolory, 2000-200 I ).

Bananas ripened at14 or 16'C in July and September all had significantly

higher levels of discolouration, compared with those ripened at 18 or 20"C.

Iængthy development in winter and ripening periods at low temperatures caused

an increase in peel discolouration, compared with that observed on bananas that

developed in warmer periods of the year, or on those that were ripened at higher

temperatures (18 or 20'C). This is due to a complex Fme-temperature

relationship, where long exposure periods to temperatures at or near the critical

chilling temperature give a similar induced injury response to that of extremely

low temperatures for short periods of time (l\4arriott, 1980; Wills et aL.,1998).

Fruit that were stressed prior to ripening and that were further sfressed when

ripened at near chilling temperatures were not able to recover from the initial

chilling exposure period. This explains the high discolouration index recorded

in bananas harvested in July and September when ripened at above chilling

temperaftres (14 and l6"C).

March harvested bananas also had higher levels of discolouration when

ripened at 14 and 16"C, compared with 18 and2}"C, even though they grew

largely during summer months, and so were not affected by chilling in the

field. However, they had been subjected to wind gusts of 125-l70lan hr-l

due to a category 2 cyclone only weeks before harvest (Bureau of

Meteorology,2000-2001), causing visual injury due to excess physical stress.

Peel appearance of March harvested fruit was also influenced by low light

84

Chapter 5: Optimisrrg Ripening Temperatures and Ethylene Concentrations Thnoughout the Year

levels due to cloud cover during late January and February, a common

occurrence in Queensland (T. Johnson, Chiquita Brands South Pacific, pers.

comm., 2002). The low light levels may also be influenced by the use of

blue bunch covers in the field, which are generally used to increase day

temperafures and reduce mechanical damage of bunches, but may also

prevent light penetration. Low light levels cause dulling of the bloom (a

waxy coating) on bananas, which has a similar appearance to mild chilling

injury synptoms on the peel (T. Johnson, Chiquita Brands South Pacific,

pers. comm.,200l).

Bananas ripened between 14 and 20'C in January, May and November all

had relatively low discolouration indexes, because their development

occurred during autumn, spring or summer when field temperatures were

above the chilling temperature of 13"C and light levels were high.

Green life, shelf life and peel discolouration was not affected by ethylene

concentrations during the initiation of ripening. This is supported by Quazi

and Freebairn (1970), who suggest the critical ethylene level required for

ripening is produced by autocatalytic ethylene production in the fruit tissues

after ripening initiation and not by the exogenous ethylene initially applied.

Ripening temperatures of 14 and 16"C and ethylene concentrations of 50

and 300 pL Ll generally resulted in higher SSC levels (l-2%) and up to a

15oá softer pulp. Similar firrnness results were reported by Rippon and

Trochoulias (1976), but were not observed by Peacock (1980). Low ripening

temperatures reduce metabolic rates and delay ripening (Wills et aL.,1998),

consequently extending the time fruit have for textural changes in the pulp as

well as for starch to sugar conversions to occur. This would only affect

softening and sugars if those en4lmes were to be affected less than visual

85


ripening (Rhodes, 1980). Even though SSC levels were significantly lower

when bananas were ripened at higher temperatures or higher ethylene

concentrations, SSC was always above 18olo, which is the lowest acceptable

level for fully ripe bananas (CSIRO, 1972). Also consumer detection of

these SSC differences would be unlikely, as people differ in their perception

and sensitivity to sweetness in various foods (Land, 1988). Also, Stevenson

et al. (1999) reported odours influence the perception of sweetness in foods,

so as banana profiles are not affected by the ripening temperatures and

ethylene levels; the SSC differences are unlikely to be detected due to the

similar banana aromas.

Weight loss occurs continually through transpiration (Turner, 1997) and so

the amount of weight loss varied, with bananas ripened at l4"C requiring the

longest ripening periods, therefore resulting in the highest weight losses. It

did not, however, affect the visual appeardnce of the fruit.

All bananas analysed had ripe banana aromas, such as fruity esters found in

fully ripe bananas (Marriott, 1980). However, my bananas also had either

unripe or over-ripe aromas at stage 6 Ripe aroma compounds (l-butanol

and 3-methylbutyl ester) and to a lesser extent unripe aroma compounds (2-

hexenal) were found to be related to the negative values of Factor 1, while

ripe (butyl esters) and overly ripe aromas (ethyl acetate) were related to the

positive values of Factor l. No off odours or reduced aroma preferences

were found due to mild chilling (Klieber and Muchu|2002), and therefore

varying ripening temperatures of l4-20oC, ethylene levels of 50-1000 pL L-t

as well as different harvesting times of the year are unlikely to have caused

aroma acceptability differences. This is in contrast with severe chilling,

where banana aroma lacks flavour and sweetness (Palmer, l97l).

86


Even though different months of the year and ripening temperatures resulted

in varying banana aroma, this aroma difference does not include unpleasant

banana aromas and so would not influence consumer acceptability. The

detection of a less ripe aroma would depend on individual consumer

preferences (Land, 1988), which for 960/o of consumers varies between

banana stage 4 (more yellow than green) and 7 (fully yellow with light

brown flecks) (Anonymous, Your guide to greater profits, undated). As

banana aroma acceptability is critical to industry, it was essential for banana

aromas to be unaffected by ripening procedures throughout the year, because

of the continual market demand.

While Peacock (1980) found that ripening temperatures between 14 and

24'C did not affect banaria weight, pulp finnness, shelf life or eating quality,

we found that ripening temperatures of bananas harvested at various times of

the year had a substantial effect on banana shelf life and visual peel

appearance. My results support Rippon and Trochoulias (1976) and Young

et al. (1932) who recommend a ripening temperature of 19"C for winter

harvested fruit. However, our findings do not support the recommendations

by Young et al. (1932) of using the higher ripening temperatures of 20 or

2l"C during summer compared to 19"C in winter.

Changes in seasonal temperatures, weather patterns and Cavendish clones

since 1932 as well as differences between the New South Wales and

Queensland growing locations may account for the slight differences in

ripening recommendations in the current study. A summer ripening

temperature of 17oC was recommended by Rippon and Trochoulias (1976)

which is similar to our recommendation. However, my results would also

support the use of ripening temperatures as low as l4"C to extend the shelf

87


life of summer harvested bananas, as long as close monitoring of field

conditions (i.e. storms, cyclones) occurs throughout the banana growing

period to avoid visual quality reduction by ripening at these low

temperatures.

5.5 Codcltlsion

Commercial ripening of bananas throughout the year at 18 or 20'C

consistently resulted in fruit with a better peel appearance than ripening at

14 or 16'C. Ripening of bananas at14 andlíoc,when fruit grew and

developed over summer, generally produced good quality fruit with extended

shelf lives, whereas bananas gro\iln during winter and ripened at 14 or l6"C

did not. However, the commercial viability of using ripening temperatures

of 14 and 16"C in summer would rely on the constant mohitoring of climatic

conditions during the banana growing period, as peel appearance can be

negatively affected by sudden climatic changes such as cyclones.

Using 50pL L-l of ethylene in commercial ripening instead of the higher

concentrations currently used, would be cost efficient without decreasing

fruit quality, although the use of higher ethylene corlcentrations of 300 or

1000 FL L-r would not be detrimental to fruit quality and shelf life.

88

Cheptes 6

6. Post-ripening: Nitrogen Atmosphere Exposure

6.1 Introduction

A consumer product survey prepared for the Aushalian Banana Grower's

Council (1999) found that if bananas had a longer shelf life, Ausfalians would

be likely to purchase more. Cunently Australians consume over 15 kg of

bananas per capita per annum (Australian Banana Grower's Council, 1999).

Bananas generally have a relatively short shelf life of only 3 days @. Charhes,

Chiquita Brands South Pacific, pers. comm .,1999),when compared to other

fruit such as apples. Banana shelf life is ended by the senescence process,

causing the visual appearance ofthe fruit peel to degrade from yellow to a

'muddy' brown colour. An extension of banana shelf life between ripening

stage 4 (more yellow than greÐ and 7 (yellow with light brown flecks)

(CSIRO, 1972) wrll appeal to consumers and result in higher purchase

quantities at one shopping occasion.

Currentþ, ripened bananas ¿ìre precooled to 13'C prior to their distribution to

supermarkets (T. Johnson, Chiquita Brands South Pacific, pers. comm., 2000)

This procedure is used to slow down fruit metabolism and therefore delay

senescence (Rippon and Trochoulias, 1976). However, this is costly, and fruit

rewarm rapidly on the shelf, thereby reducing shelf life.

89

Chapter 6: Post-rþening: NiÍogen Atmosphere Exposure

Previous research has focussed on ways to extend banana green life. For

example, modified aÍnosphere packagtng has been suggested as a substitute to

low temperature storage (Scott et a1.,1970); however, this storage is costþ as it

requires ethylene absorbent packaging and carefü handling to prevent damage

to bags and consequently their inner atrnosphere. Wills ¿/ al. (1990)

successfully used short-term nitrogen treafinents to increase the green life (from

harvest to yellow with green tips) of bananas, with nitrogen atmospheres of 3

day duration applied immediately after harvest, increasing their green life by

42%. Nitrogen atmospheres will reduce the availability of oxygen to the fruit

and will in turn inhibit ethylene production and ripening (Thompson, 1998) in

an inexpensive and easily applied method.

A method for slowing the rate of senescence in ripening bananas to extend

shelf life from the point of sale to consumption is required. The aim of this

research was to examine whether storing fruit temporarily in nihogen

atmospheres after partial ripening could extend the sheHlife of bananas without

affecting their appearance or aroma. Results from this study have been

published by Klieber et al. (2002, see attached publications).


6.2.1 Plant material and preparation

All fruit was harvested and prepared as described in chapter 3. After

preparation, bananas were randomly allocated to l0-L plastic ripening

containers in the presence of a carbon dioxide scavenger (160 g of Ca(OÐz)

and using a saflrated KNO3 solution to maintain 95o/orelative humidity. All

fruit were ripened at l6"C with 300 pL L1 ethylene gas on two consecutive days

and ventilated daily. At ripening stage 3 (more green than yellow) or 4 (more

90

Chapter 6: Post-ripening: Nitrogen Atmosphere Exposure

yellow than green) fruit were held for various periods in nitrogen (l{z) or

ripened nonnally at room temperature (2lo/o, at22"C).

6.2.2 Nitrogen application for various periods of time at different

ripening stages

Bananas at ripening stage 3 or 4 were held in nitrogen atmospheres (0%

oxygen) for 0, 6, 12 or24 hrs. The two control (0 hrÐ treatments were ripened

normally in air until ripening stage 3 or 4, then removed from the plastic

containers, placed into unsealed polyethylene bags and ftuther ripened at22'C

until the end of the trial. Nitrogen treated bananas were placed in containers

that were flushed with high puntyN2 (BOC gases, Adelaide) for five min.,

before being sealed for the allotted treaûnent periods of 6,12 or 24 hrs at22C.

Afnosphere samples were taken from sealed containers and analysed using a

thermal conductivity detector gas chromatograph (Varian 3300, Varian

Associates Inc., Mulgrave, Victoria) to ensure oxygen (O) and carbon dioxide

(COt levels were ( lo/o and 0% (Wills et al., 1990), respectively, as explained

in section 3.4. On completion of treaünents, fruit were placed in vented

polyethylene bags at22"C to complete ripening.

6.2.3 Nitrogen atmosphere storage in the presence of low or high

relative humidity

A second nitrogen atrnosphere experiment was performed to observe whether

humidity stress was a factor in the results obtained from the previous

experiment. Bananas were prepiled as above and held continuously in air in

folded polyethylene bags (95% RIÐ or were pre-treated in nifrogen for 24hrs.

The atmospheric composition was again monitored. Nitrogen pre-treated

9l


bananas were then subdivided into two lots; one was held in open polyethylene

bags (65o/oRII) or in open bags with saturated KNO3 solutions added(95%

RII). The relative humidity inside polyethylene bags was measured using a

hand held humidity probe (IIM34C, Vaisala, Helsinki).

6.2.4 Assessments

Atreafnent unit consisted of 12 fruit, six of which were used to assess external

characteristics of the fruit and the ¡srnaining six were used to assess intemal

properties of the bananas. All bananas were assessed for weight loss, shelf life,

discolouration, ftrmness, SSC and aroma as described in sections 3.5 and 3.6.

Statistically, the first experiment was set up as a 4x2factonal randomised

complete block design with 3 replicates. Treatnents included the effect of 0

(air storage) , 6, 12 or 24 hrs of nitrogen storage applied to bananas at ripening

stage 3 (more green than yellow) or 4 (more yellow than green). Replicates of

the experiment were conducted in 3 consecutive weeks on bananas that were

randomly allocated to all freatments.

The second experiment was established as a 3 x2 randomised complete block

design with three replicates and two humidity treafinent levels. Each replicate

consisted of the following nitrogen storage treatnents, 0 (air storage),24hrs

with a high hunidity and24 hrs with a low humidity. All experimental units

were randomly distributed to treatments.

Analysis of most data (shelf life, SSC, firmness and discolouration) was

conducted using the General ANOVA directive in Genstat 5, @elease 41,4ú

edition, 1998, Lawes Agricultural Trust, Rothamsted). Shelf life data for the

first experiment was squared, but has been transformed into original data units

92

Chapter 6: Post-rþening: Nitrogen Atmosphere Expostue

for Table 6.1. Treafnent effects were examined for significance using a least

significant difference test (-SD) at the 5olo level.

Finally aroma data was analysed by principal component anaþis @CA) using

the Pirouette analysis package (Infometrix Inc, Woodenville, WA). Each model

was validated using a cross validation method, which indicated the principal

components that totalled greater than 80% of the variation. Fruit with similar

volatile compounds were grouped together and separated from fruit with

different volatiles on plots.

6.3 Results

6.3.1 Nitrogen application for various pefiods of time at different

ripening stages

Bananas stored in nifiogen atmospheres at ripening stages 3 or 4 for 6,12,24

hrs had similar shelf lives and SSC readings to bananas ripened in air (Table

6.1). The amount of discolouration observed on banana peels varied

significantly with the nitrogen atrnosphere applied (Table 6.1). Nitrogen

exposed bananas had large patches of brown discolouration, which were not

present oh bananas ripened in air. However, air-ripened bananas did have the

appearance of brown vascular sfieaking similar to that found on chilled fruit and

the appearance of the brown discolouration patches on nitrogen-exposed fruit

increased with the length of nitogen exposure (Figure 6.1).

93

Chapter 6: Post-rþening: Nitrogen Atmosphere Exposwe


after exposure to a nitrogen atmosphere for 0, 6, 12 or 24 hrs at ripening stage 3

or 4 at22"C.

Ripening stage and period of time (hrs) for which bana¡ras were stored mnitrogerf

Banana

assessment

Ripening stage 3 Riperung stage 4

0 6 1224067224

Shelf lfe(days)t

Firmness(kPa)-

Solublesolidcontent (%)

Weight loss(%)

Discolourlncex

l0l a 104 b

23.6a 23.0a 23.2a 23.3a 23.2a 23.5a 23.3a 23.1a

2.0 a 2.O a 2.1& 2.2 c 2.1 ab 2.1 ab 2.O ab 2.1b

7.4a 7.4a 6.7a 7.3a 7.3a 7.6a 7.2a 7.6a

0.0 a l.4b 20 c 2.7 d 0.0 a 1.4b 7.9 c 2.8 d

'Dataare averages of 3 replicates and different superscripts within arow denote

significant differences at P< 0.05 using a LSD test.

v Average shelf life (dayÐ data has been back transformed from squared

analysis data.

* Only ripening stages are significarft at the 5Yo level, therefore the averagefirmness of bananas stored at ripening stages 3 or 4 were the only averagesglven.

* Discolouration index where, (0) healthy fruit, (1) slight, (2) mild, (3) severe

discolouration.

94


E

Figure 6.1 : Brown discolouration observed on nitrogen treated Cavendish

bananas cv. Williams increased with exposure periods; (A) air ripened, (B) 6,

(C) 12 and (D) 24fus of nitrogen exposure.

95

Chapter 6: Post-ripening: Nitrogen Atmosphere Exposwe

Bananas exposed to nitrogen or air at ripening stage 3 hada3Yo lower pulp

firmness than those exposed at ripening stage 4 (Table 6.1). However, pulp

firmness was similar for all bananas from one ripening stage. Nitrogen

aÍnospheres also had a significant effect on weight loss of bananas stored at

stage 3, with longer periods of nitrogen exposure resulting in slightly higher

weight loss. However, this was not observed for fruitremoved at colour stage 4

(Table 6.1).

Bananas at ripening stage 6 had different aroma characteristics after various

periods of air and nitrogen storage. The PCA models built to compare mass

spectrometric chemical nose data of all nitrogen exposed as well as air ripened

bananas explained glYo of the total variance present with 2 factors; as 85% of

the total variance related only to Factor 1, so too will the focus of the results.

Score plots with the 2 factor elements showed discrimination between nitrogen

held and air-stored fruit (Figure 6.2). However, some nitrogen exposed fruit had

a central value disfibution within the plot (eg. C, Figure 6.2), while some (eg.

D) were located to the left of the plot. This zuggests some variability between

nitrogen treated fruit, even though no clear fend for exposure period was

apparent.

96


raêJÉr--e\

'E.Ë'ËE\/

1!oõlf

It\

a'l'1"

Fadorl

Figure 6.2: The score plot of ripe Cavendish bananas cv . Williamr exposed to

nitrogen atrnospheres for 0 hrs (air) at stage 3 or 4 (,\ g, - ) at stage 3 for 6,

l2,24hrs (C, D, E, - -) or at stage 4 for 6,12 or 24hrs (F, G, II, ..." )

Observations between the plot of the mass to charge ratios of ions (variables)

that contribute to sample discrimination @igure 6.3) and the sample positions in

the score plot @igure 6.2) are evident, with a strong correlation between ion 55

and air stored bananas, while nitogen held fruit largely correlated to ion 6l.

10

97

Chapter 6: Post-ripening: Niüogen Atmosphere Exposwe

ôt

f;

F#

Figure 6.3: The le¿dings plot of ions (variables) derived from ripe, nitrogen

exposed Cavendish bananas cv. William,s at ripening stage 3 or 4 for 0,6,12 or

24tus.

98


6.3.2 Nitrogen atmosphere exposure in the presence of low or high

relative humidity

Nitrogen ahospheres with high or lowhumidity and conftol (air) treatments

resulted in similar banana shelf life, pulp flrmness, SSC and weight loss when

ripe. Low Oz injury appeared equally on bananas exposed to low or high

relative humidity, but not in the control treatment (Table 6.2).


after being ripened at ripening stage 4 in air or nitrogen atmospheres for 24 hrs

with a beaker of water or without a beaker of water at22"C.

Banana assessment

at ripening stage 6

Control

(Air,95%RII)

24hrN2 storage Iwater(95%oRÐ

24hr N2 storage -water (657oRÐ

Shelf life (davÐ

Firmness (kPa)

Soluble solid

concentration (o/o)

Weightloss (7o)

Dscolouration

index

7.0 a

104 a

24.2 a

1.7 a

0.0a

8.0 a

103 a

23.4 a

2.0 a

1.1b

7.4 a

102 a

24.0 a

1.8 a

1.4 b

' Data are averages of 3 replicates and different superscripts within a row

denote signiflrcant differences at P< 0.05 using a LSD test.

99

Chapter 6: Post-rþening: Nifiogen Atmosphere Exposure

The PCA model explainedST%o of the total variance among featments with 2

factors; both Factors I and 2 willbe observed in these results as both contribute

alargeproportiontothetotalvariance(Frgure6.4). Factor I consisted of57o/o

of the total variance with distinct discriminating aroma gtoupings, with one

cluster containing bananas ripened in air as well as those exposed to nifogen

with a low humidity and the other cluster consisting of bananas held in nitrogen

with ahighhumidity.

10

Facbrl

Figure 6.4: A score plot of ripe Cavendish bananas cv . Williams exposed to

(4, -;

nifiogen for 0 hrs (air), (8, -

) nitrogen for 24 hrs in a low humidity

or (C, -)

niúogen for 24 hrs in a high humidity.

c{ooõL

'cU

j$.

c c

E

c

c

'dc

'o;%

100

Chapter 6: Post-ripening: Ninogen Atmosphere Exposure

The cluster containing air-ripened bananas as \Mell as nitrogen atrnosphere held

bananas in a low humidity were closely associated to the ion with a mass to

charge ratio of 56 (Figufe 6.5). Niüogen exposedbananas in ahigh humidity

produced aromas that clustered around the ion with a mass to charge ratio of 70

Factor 2 consisted of 30o/o of the total variance. Bananas held in nitrogen in a

high or low humidity were related to ions with a mass to charge of 61 and 55,

whereas air ripened bananas were generally only associated with 55 (Figure

6.s).

o

soñL 88

R7

87'71

Facbr,|

Figure 6.5: The loadings plot of ions (variables) for ripe Cavendish bananas cv

Williams exposed to nitrogen for 0 hrs (nr),24 hrs in a low humidity or for 24

hrs in a high humidity.

l0l

Chapter 6: Post-ripening: Nihogen Atmosphere Exposure

6.4 Discussion

Short-term nitrogen exposure after ethylene initíation did not slow fruit

deterioration or extend banana shelf life, and so further research into post

ripening treatrnents to extend shelf life is required.

Liu (1976ab) found ethylene initiatedbananas stored nalo/o 02 atrnosphere at

14"C ripened soon after beingfiansferred to air at 21"C. These findings support

the observation that storage of ethylene treated produce in oxygen depleted

atmospheres leads to a faster rate of ethylene production and therefore ripening

upon returning to a normal (air) afnosphere, than fruit held only in aerobic

conditions (Wang et aL.,1990).

This increased rate of ethylene production after the anaerobic nifrogen

treatnent causes an acceleration of ripening after the nitrogen treahnent, so that

nitrogen treated fruit have a similar shelf life to that of air-stored fruit.

However, this response was conüadictory to that shown in breaker tomatoes,

where nitrogen treaûnents delayed ripening by increasing intemal ethanol and

acetaldehyde production via anaerobic respiration, without causing any

discolouration to the fruit @esis and Marinansþ 1993; Ratanachinakom et al.,

1997). Anaerobic atmospheres may have formed in the pulp, but bananas may

not be as sensitive to ethanol (refer to ethanol chapter) or are able to

remetabolise ethanol faster than tomatoes and so the anaerobic effect was not

apparent.

Short-terrn nitrogen storage did, however, cause dark discolouration on the peel

of fruit. Similar discolouration has previously been observed on bananas stored

between 5 attdlfYo CO2(Thompson, 1998). Thompson (1998) compiled data

102

.-- :l

Chapter 6: Post-ripening: Nitrogen Atmospherg Pxpqqllg

from a number of sources and foturd that levels of < lo/o 02 in bananas exposed

for an unspecified time period cause low Oz injury symptoms such as dull

yellow or brown peel discolouration, failure to ripen as well as 'ofP aromas.

Wills e/ at. (1990) also found some discolouration on nitrogen treated green

fruit. Brown peel discolowation was observed in this investigation on bananas

that had been stored in nifiogen for 6 hrs and the appearance of the

discolouration increased progressively as the storage period in nitogen

increased. Therefore, the skin discolouration on bananas stored in nitrogen

atmospheres for 6 hrs or longer probably was due to the low 02 stress in the peel

damagtng cellular membranes and allowing the en4matic browning reaction to

occur

Even though there was a statistically significant difference between the pulp

firmness of bananas held in niüogen atmospheres at ripening stage 3 and 4 this

difference would probably not be detected by consumers during consumption,

as most consumers eat bananas ranglng between ripening stage 4 andT

(Anonyrnous, Your guide to greater profits, undated) which vary in firmness.

All SSC readings reported in the studywere above the 78Yo level (when fully

yellow) set by CSIRO (1972) guidelines. There were only slight differences

between total weight loss readings measured between nitrogen exposed and air-

ripened fu:/rt (2.0-2.1%o) astranspirational losses during ripening are part of the

normal ripening process @almer, l97I).

The chemical nose was used as Ím easy, quiclq reliable andrepeatable method

of aroma analysis as opposed to human nose detection, which tends to be time

consuming and may have been exhausting for human judges due to continuous

and multiple aroma sampling (Muchui, 2000).

103

Chapter 6: Post-rþening: Nitrogen Atmosphere Exposure

In the first experiment, Factor I showed most of the bananas exposed to

nifiogen aûnospheres for the various periods of time differed in aroma

compared to control bananas stored in air. Bananas stored in air were strongly

correlated to the ion with a mass to charge ratio of 55, which arise from the

known banana aroma compounds 3-methylbutyl ester and l-butanol. Pulp from

bananas that were stored in nitrogen were mainly correlated to an ion with a

mass to charge ratio of 61, which arises from ethyl acetate, a banana aroma

compound that has an over ripe aroma character. The fruit in Figure 6.2 that

clustered in between the extremes of ion 55 and ion 6l had amixed aroma of

overripe and ripe tones.

The presence of ethyl acetate at stage 6 in nitogen-stored bananas may have

been due to anaerobic conditions, because in normal ripening the production of

ethyl acetate generally only occurs in late Senescence or when bananas are

considered inedible (Macku and Jennings, 1987). As ethyl acetate was not

present in control fruit at the snme ripening stage, it was likely to be accelerated

by the nitrogen exposure period. Initiation of anaerobic respiration in tissues

and the consequent triggering of fermentation reactions leads to the production

of off-odours in fruit (Wills et a1.,1998), like the over-ripe aroma identified in

the results. Time fruit spent in anaerobic conditions would have influenced the

production of over-ripe aromas, and this would account for the mixed aromas

produced by some nifogen-stored fruit. Even though the aroma results were

obtained when banana peel was of stage 6 appearance, the nitrogen afnosphere

treaÍnent may have triggered a stress reqponse resulting in ær increased

production of over-ripe aroma compounds.

In the second experiment, aroma data varied more due to the humidity factor

than exposure to nifiogen. Therefore, Factor I showed differences in aroma

704

Chapter 6: Post-rþening: Nitrogen Atmosphere Exposure

profile caused by the humidity factor, whereas the impact of nitrogen exposure

on aroma was observed through Factor 2. Interestingly, Factor I and2 appeared

in the reverse compared to the previous experiment, that is Factor I became

Factor 2 when considering the ions that contributed to each factor.

Air-ripened and nitrogen held bananas in low humidity had aromas related to

the ion with a mass to charge ratio of 56 associated with the known banana

compound l-hexanol that gives an unripe green tone. Unlike those held in

nifrogen with high humidity that had aromas relating to ion 70 that is largely

associated to ripe aroma profiles and compounds like 3-methylbutyl ester, 1-

butanol and propanoic acid; these volatile compounds are also strongly

correlated to ion 55. Bananas held in nitrogen in high or low humidities were

also related to both ions 61 and 55, while air-ripened bananas were only further

associated with ion 55.

Ion 6l is present in the volatile compound ethyl acetate, which is known to be

attributedto overripe banana arom4 whereas ion 55 is identified in compounds

like 3-methylbutyl ester and l-butanol that are associated with ripe aroma

profiles. Ions 55 and70 are both highly correlated to ripe aroma compounds

like 3-methylbutyl ester and 1-butanol, but ion 55 is also associated with 2-

hexenal, while ion 70 is collectively associated with ethyl acetate. Therefore,

even though the ions with a mass to charge ratio of 55 and 70 are predominantly

associated with ripe aroma compounds, they are also correlated to a lesser

extent with unripe or overripe aromas, respectively.

Therefore, the aroma profiles for this second nitrogen experiment consisted of

air ripened bananas having an unripe or green aroma with some ripe tones.

Nitrogen exposed bananas in low humidity also had an unripe green aroma but

105


also had both ripe and overripe tones present. Finally nitrogen held bananas in

high hurnidrty had a ripe to ovempe aroma.

I¡sses in aroma volatiles have been reported to occur in climacteric fruit when

held in low relative humidities (Grierson and Wardowski,l9TS). The water

stress can cause changes in the strucfire and conf,rguration of en4rmes and as a

result en4me activities charrge, adversely affecting metabolic rates and other

chemical pfocesses such as aroma production (Noggle and Fritz, 1983).

Over-ripe volatiles corfélated sirorrgly with bahanas stored in nitrogen in a high

or low humidity; this can againbe related to anaerobic respiration accelerating

the pfoduction of alternative aroma compounds. The production of over-ripe

volatiles at ripening stage 6 (fullyyellowpeel), instead of at ripeningstageT

(yellow with light brown flecks), when over-ripe volatiles are generally

expected, suggests that an off-odour may have formed as a result of nitrogen-

storage and the stress it caused. The fermentative production of over-ripe

volatiles during anaerobiosis is similar to the aromas commonly produced

during senescence, where cell walls start to be degraded, leading to a reduction

in gas exchange in fruit (TaizandZniga,1998). This causes localised

anaerobiosis in bananas as gas diffiision throughout the fruit decreases with

increased resistance to gas exchange @erez and Beaudry, 1998).

6.5 Conclusion

Nitrogen abnosphere exposure after ethylene initiation prior to commercial

marketing of bananas is not a viable postharvest option to extend shelf life. The

exposure caused peel browning without delaying ripening or senescence.

Aroma compowrds were also found to differ among air-stored and nitrogen-held

106

Chapter 6: Post-rþening: Niüogen Atmosphere Exposure

bananas when fully ripe, but these differences did not necessmily have an

adverse effect on flavour, as aromas produced are all naturaþ produced in

bananas at some stage during ripening. Therefore, iloma acceptability would

depend on the consumer's individual aroma preference. Overall it was

concluded that niüogen aÍnosphere storage is not an effective method for

extending the shelf life of ripening bananás and filrther research on alternative

treahnents is required.

to1

Chnfter 7

7. Ethanol Atmosnhere Exposure

7.1 Introduction

Appropriate handling and management of bananas is essential during

distribution, as bananas ripen and senesce quickly after initiation with ethylene

(Marriott, 1980; Israeli and Lahav, 1986). Methods of slowing the onset of

ripening of bananas prior to initiation have been researched using controlled

atmospheres (Wills et a\.,1982) or nitrogen aûnosphere storage (Wllls et al.,

1990). These types of storage conditions induce anaerobic metabolism and

ethanol production (Pesis and Avissar, 1989; Pesis and Marinansky, 1993),

which prolongs the storage of pre-climacteric fruit. However, industry also

requires techniques to slow senescence of ripe bananas to satisfii consumer

demands of a shelf life longer than 3 or 4 days (T. Johnson, Chiquita Brands

South Pacific, pers. coÍìm., 2000). Very low oxygen afnospheres for short

periods of time after ethylene initiation of ripening has been reported in chapter

6, but this post-ripening storage treatnent did not extend banana shelf life and

promoted discolouration on fruit.

Drect application of ethanol has been suggested as a possible alternative

rnethod to disrupt the ripening and senescence processes of climacteric fruit

(Salweit, 1989; Beaulieu and Saltveit, 1997; Podd and Van Staden, 1998), and

108

Chapter 7: Post-ripening: Ethanol Atmosphere Exposure

of delaying banana ripening (Ilewage et al., 199 5 ; Ritenour et al., 1997 ).

However, banana ripening was not delayed in the latter studies in which ethanol

was exogenously applied on soaked filter paper in a sealed container; neither

study reported whether bananas actually absorbed ethanol. A possible way to

force ethanol into bananas is via vacuum infiltration, a technique effectively

used by Ratanachinakorn et al. (1999) on tomatoes and so vacuum infiltation

of ethanol was examined in this study, to extend banana shelf life.

The first objective of this study was to determine whether vacuum infiltration

of ripening initiated bananas with ethanol atmospheres could prolong banana

ripening and shelf life without affecting quahty. Secondly, the studywas

performed to show the effect of vacuum infiltration on the absorption of ethanol

by fruit that have thick skins, like bananas.

7.2 Matenals and methods

7.2.1 Plant materlal and preparation

Bananas (Cavendish cv. Williams) were harvested twice during September

2001, from a single plantation in Tully, Northern Queensland. Theywere

transported and prepared using the procedure described in section 3.1 and3.2.

All bananas were labelled and randomly assigned to five treatment groups each

containing 18 bananas.

Ripeningwas initiated at 18"C atg1Yorelative humidityin an atmosphere of

300 pL L I ethylene gas in the presence of a carbon dioxide scavenger (160 g of

Ca(OII)2per container). Containers were vented after 24 hrs ærd ethylene

reapplied.

109

Chapter 7: Post-rþening: Ethanol Atmosphere Exposure

7.2.2 Determination of ethanol volume

The ethanol volume neededto provide a saturated ethanol atmosphere was

measured byplacing 1, 0.5, 0.25, 0.185, 0.05, 0.015 mL of absolute ethanol into

a 10-L vacuum desiccator with 6 bananas. The desiccator was evacuated to 10

kPa pressure for 5 min. and then repressurised with bananas left in the

atmosphere for 1 or 30 min. After this time the volume of ethanol liquid left in

the bottom of the desiccator was obsered. For each volume of ethanol tested

residual ethanol was present, suggesting that suffrcient ethanol was present to

allow the atmosphere to become saturated. In frrther experiments I mI of

ethanol was used.

7.23 Effect of ethanol vapour and holding times after repressurising on

the quality and ripening of bananas

To determine the relationship between pressure and volume of air removed

frorn bananas, pres$ile was reduced to70,40 or l0 kPa as described by

Ratanachinakorn (2000). The amount of air expelled from bananas was

measured by submerging individual bananas in water in a 10-L desiccator. The

banana was covered with a funnel corurected to a graduated measuring cylinder

filled with water before desiccator evacuation at the various pressures for 5 min.

@igure 7.1). Air evacuated from the fruit replaced the water in the cylinder and

its volume was thus measured.

ll0


Figure 7.|:Dtagran of air evacuation set up, consisting of (Ð 10-L glass

desiccator, @) vacuurn pump, (C ) measuring cylinder and (D) Funnel (covering

a single banana).

The effect pressure had on visual appe¿ìrance (discolouration), shelf life and

weigþt loss of badanas was also observed. Apreliminary studywas performed

where 2 replicates of 6 fruit were ripened normally (in air) or vacuum treated in

air for 5 min. at 10, 40 or 70 kPa pressure after 48 hrs of norrnal ripening at

l8"C with 300 ¡rL Lr ethylene gas on 2 consecutive days. After vacuum

infiltration the treated bananas were held in the aÍnosphere for 30 min., then

placed into polyethylene bags at20"C where they were ventilated daily until

assessment as described in section 3.5. Results showed no significant

differences between shelf life, discolouration or weight loss of bananas ripened

nonnally or vacuum infiltrated. Therefore 10 kPa pressure, the maximum

lll


vacuum, was used in the following ethanol vacuum infiltration experiments

7.2.4 Ãpplication of ethanol atmosphere

After 48 hrs of ripening, bananas were removed from their containers and

vacuum infilfiated in a 10-L desiccator at 10 kPa. The vessel contained air only

or 1 mL of absolute ethanol in a Petri dish @gure 7 .2). The vacuum was

applied for 5 min., then the vessel was repressurised and kept in the atrnosphere

for a further 10 min. or 3 hrs at22"C. After the treatment period, bananas were

assessed and the remainder allowed to continue ripening at 18"C until they

reached ripening stage 4 (more yellow than green, CSIRO, 1972). They were

then placed into polyethylene bags and ripened at22"C until final assessments at

stage 6 or 7.

112


Figure 7.2: Vacuum (A) infrlfiation of ethanol atrrosphere generatedfrom 1

mL absolute ethanol (B) in a l}-L glass desiccator (C ) into 6 ripening initiated

bananas (D), in the presence of 160 g Ca(OÐz@).

7.2.5 Y olatile analysis

Banana peel and pulp were analysed for ethanol concentrations immediately

after treatment application, as well as after ripening at colour stage 6. Peel and

pulp samples were excised from the middle of each of the treated fruit, using the

method described by Ratanachinakom et al. (1999). Excised tissues were cut

into 5 mm cubes, with I g sænples of both peel and pulp being separately frozpn

in liquid N2 The samples were then placed into pre-cooled 5 mT. capped test-

tubes and stored at -18 to -20"C until analysis.

Ethanol analysis began with the thawing of the frozpntissue samples at0-2C

for 60 min., after which theywere heated at 30'C for a fiuther 60 min. One mL

113


headspace samples were u/ithdrawn from the test-tubes and analysed using

flame ionisation gas chromatogaphy (FID-CC, Varian 3400. Varian Australia,

Mulgrave, Victoria), with a stainless steel colunn (1.8 m x 3.2 mm OD) packed

with Gas Chrom@ 254 of 801100 mesh. The column temperahre was set at

l40oc, with injector and detector temperatures at 150"C and 160oC,

respectively. Flow rates of air, hydrogen and the carrier gas nitrogen were 300,

40 and 50 mL min.-l, respectively. The volatilisable tissue content of ethanol

was expressed as nL g.f.\ú.-I.

The ethanol stærdard used to calibrate the GC wÍrs prepiled according to Shaw

et al. (1991) and Ratanachinakorn (2000). Five pL of analytical grade ethanol

was precooled at -18"C due to its volatility and injected into a sealed 2 L flask

that had previously been preflushed for 5 min. \ 'ith high purity nitrogen and

contained a magnetic stirrer. The gases were mixed with the magnetic stirrer at

20"C for 60 min. before being used to calibrate the GC.

7.2.6 Banana and statistical assessments

Banana shelf life and peel discolouration was visually assessed, while pulp

firmness, SSC and weight loss were also assessed as detailed previously in

sections 3.5 and 3.6.

Each of the September hmvests were considered as one of two experimental

replicates. The experimental replicates consisted of five treatment groups,

where bananas were normallyripened in air (control), vacuum infrlrated with

air or over lml- of absolute ethanol, with the latter two freatments kept in the

atrnosphere for l0 min. or 3 hrs after vacuum infiltration.

All treafinent groups consisted of l8 banana fingers, where six fingers were

1,t4


used for shelf life and discolouration assessments, six fingers for firmness, SSC,

weight loss and ethanol concentration measurements at stage 6 and the final six

fingers for ethanol concentration analysis immediately after treatnents. Only

six bananas from a single treatment could be vacurÍn infiltated at one time, so

that bananas for different assessments had to be prepared on different days. The

fruit that were held for experimental preparation on different days were stored at

14oC, similar to bananas stored in industryprior to ripening initiation (T.

Johnson, Chiquita Brands South Pacific, pers. comm., 2000). All data were

analysed using general analysis ofvariance and a least significant difference test

(LSD) atthe 5%olevel, using the Genstat 5 program (Release 4.1.,4h edn., 1998,

I¿wes Agncultural Trust, Rothamsted Experimental Station).

7.3 Results

Bananas that were vacuum infilffated and exposed to ethanol atmospheres did

not have an extended shelf life, but ripened similarly to those ripened with or

without vacuum infiltration in air (Table 7.1). Firmness, SSC and discolouration

were similar for all treatments at about 110 kPa, 24% and 1.5, respectively, but

weight loss did vary between treatrnents with those that were vacuum infiltrated

losing less weight than bananas ripened without vacuum expo$ne.

lls

Chapter 7: Post-rþening: Ethanol Atmosphere Exposwe

Table 7.1: Ripe characteristics of Cavendish bananas cv. Williams after

ripening with ethylene (300 pL Ll) at 18oC, treated by vacuum infilfiation over

air or ethanol and held in the resulting atmosphere for 10 min. or 3 hrs. After

the holding period the bananas were held at 18'C until they reached stage 4 and

then were ripened at22'C until assessed.

Vacuum treaf,nen(

No

vacuum

Vacuum

+ l0min.

holding

Vacuum +

3hrs

holding

Vacuum

overEtOH

+ 10min.

holding

Vacuum

overEtOH

+ 3hrs

holding

Shelf life

(davÐ

Firmness

(kPa)

Soluble

solid

concent (%)

Discolour.

index

Weight loss

(%)

ll2a 107 a lll a

' Dataare averages of 2 replicates where different superscripts within arow

denote significant differences at P< 0.05 using aLSD test.

3.8 a 4.3 a 3.8 a 4.2 a 4.4 a

110 a ll3 a

245 a 23.9 a 24.5 a 24.I a 24.5 a

1.8 a 1.6 a 1.4 a 1.3 a 1.4 a

2.3 c 1.9 ab 2.0b 1.8 a 1.8 a

i16


Banana peel and pulp ethanol concenftations, measured immediately after

vacunrn feaûnents as well as at rþening stage 6 are shown nTable7.2.

Ethanol concentrations were seven and 40 times higher at 78 and 473 rtL g.f.

wt.-t, in the peel of bananas held for l0 min. and 3 hrs in ethanol, respectively,

compared to concentrations in the peel of bananas ripened normally or vacuum

infiltrated with air. Pulp ethanol concentrations of bananas measured

immediately after vacuum infiltration with ethanol were similar to those ripened

normally and those vacuum infiltrated with afu. Also, ethanol concentations of

banahas held in ethanol ahnospheres were significantþ higher in the peel tissue

compared to the pulp tissue of the bananas measured immediately after their

treatrnent. At colow stage 6, ethanol concenfiations of pulp tissues were higher

than those found in peel tissues for all banana treatnents, and all were higher

conrpared to levels immediately after treaünent. Ethanol levels in the peel

reached 547 íl-g. f. !vt.-t and in the pulp 778 nl'g. f. wt.-t.

tt7


Tabte 7.2zPeel and pulp tissue ethanol concentations of Cavendish bananas cv

Williams after ripening with ethylene (300 pL L 1) at 18oC, teated by vacuum

infiltration over air or ethanol and held in the resulting atmosphere for l0 min.

or 3 hrs. After the holding period, bananas were held at l8"C until they reached

stage 4 andripened at22'C until assessed.

EtOH concentration (nL g.f.wt.-])'

Immediately after vacuum

treaûnent

At ripening stage 6 Y

Vacuum

treaûnents

Peel tissue Pulp tissue Peel tissue Pulp tissue

No vacuum

Vacuum + held

for 10min.

Vacuum + held for

3hrs

Vacuum overEtOH

+ held for 10min.

Vacuum over

EtOH + held for

3hrs

l0a

lla

4a

78b

413 c

14a

14a

13a

13a

2la

699 a

525 a

953 a

800 a

484 a 676 a

454 a 695 a

573 a 767 a

' Dataare averages of 2 replicates where different superscripts in tissue

columns denote significant differences at P< 0.05 using a LSD test.

ll8


7.4 Discussion

After ripening initiation, fruitwere vacuum infilfiated over either air or ethanol,

with or without prolonged holding periods, but banana shelf life was not

affected. Firmness, SSC and discolouration were also similar and in the norrnal

range for colour stage 6 bananas harvested after awinter growing period (as

observed in section 5.4). Even though weight loss results were variable between

vacuum and non vacurrrn featments, the overall effect on banana appearance

would be minimal, as banana senescence is identified by consumers as over

maturityrather than weight loss or shrinkage (Grierson and Wardowski, 1978).

All bananas ripened similarly, with exogenously applied ethylene initiating the

banarìa ripening process followed by autocata$ic endogenous ethylene

production (Seyrnour, 1993; Oetiker and Y*g, 1995). Contaryto myfindings

with bananas, Saltveit (1989) using tomatoes reported that ethanol vapour

application delayed mature green tomatoes ripening by delayng ethylene

production. A possible reason for the ineffectiveness of ethanol vapour on

banana ripening is that acetaldehyde (AA) not ethanol is the active component

in inhibiting ripening (Beaulieu et al.,1997;' Beaulieu and Saltveit,1997;

Ritenour et al., 1997} If ripening initiated bananas had insufficient alcohol

dehydrogenase (ADIII activity to metabolise ethanol into physiologlcally active

levels of acetaldehyde, ripening would not slow @itenour et al.,1997).

However, this is not the case as ripening initiated bananas do have suffrcient

ADH activity at stage 2 (greenwith traces of yellow) of 0.5 ¡rmol min.-l g t that

increases each day during banana ripening to 1.2 pmol min.-r gl by stage 7

(Hyodo et al. , 1983; Seymotn, 1993) . In tomatoes ADH ranges from I .5 - 4

pmol min.-lgl lRatanachinakorn, 2000). Arecent studyperformed on breaker

tomatoes (Beaulieu and Salweit,1997) found that climacteric fruit tissues after

ll9


ripening initiation were sensitive to ripening inhibition by ethanol

While vacurÍn infiltration with or without prolongedholding forced ethanol

absorption into the banana peel, it is unlikely ethanol infiltrated into pulp tissue,

due to the thickness of banana peels. Unlike tomatoes, where ethanol was able

to diffuse readily through the tomato stem scar and skin into the pericarp and gel

of the tomatoes (Ratanachinakorn et a1.,1999). This failure to penefrate into

pulp tissue was critical in the inability of ethanol to slow the ripening process in

bananas, becáuse pulp tissues play an irtrportant role in ethylene production and

consequently through internal ethylene diffusion into the peel (Vendrell and

McGlasson ,l97l). Peel degreerting is regulated by pulp riperling and ethylene

production (Vendrell and McGlasson, 1971), which was not inhibited by the

ethanol treatment, as vacuum infiltration did not force ethanol into the pulp.

Consequently banana peel colour did not respond to applied ethanol @itenour

et aL.,7997).

Peel and pulp ethanol concentrations were measured immediately after bananas

were treated and were initially low, except for banana peel that had been

infiltrated and held over ethanol for 10 min. or 3 hrs. However, peel ethanol

concentrations were comparable for all batranas when fully ripe and were higher

than immediately after treatrnent. A similar trend was found for the pulp, but

pulp ethanol concentrations \ryere significantþ higher than in the peel when

bananas were fully ripe. The increase in peel and pulp ethanol levels in ripe

fruit was observed in all bananas and can be attributed to the production of

ethanol during the later stages of ripening (Marriott, 1980; Hyodo et aL,1983;

Seymour, I 993 ; Pesis , 1996). My results support the previous studies that found

ripening climacteric fruit like bananas have initiallylow ethanol concentrations

which increase during the post-climacteric phase. The normally high ethanol

120


levels during ripening may also explain that the high peel ethanol levels did not

influence ripening. It is also possible that ethanol declined initially after

treatnents due to re-metabolism or volatilisation @esis and Avissar, 1989), thus

allowing fruit to start ripening at relatively low ethanol levels that increase as

ripening progresses.

7.5 Co[cluslon

In sünmary, vacuum infiltration with ethanol did not delay ripening of

banahas, in contrast to the situation in tomatoes that take up ethanol into the

pulp through the stem scaf 4fld the skin. There is scope, however, for testing

other ethanol increásing methods that affect the pulp, such as ntodified

atrrrosphetes.

721

Ch"pt''br I

8. Post-ripening: l-Methvlcvcloproþene Exposure

8.1 Introduction

The distinct ripening pattern of bananas ensures that once ripening has been

initiated, subsequent deterioration is rapid, which is of concem to marketers and

conswners alike (T. Johnson, Chiquita Brands South Pacific, pers. coÍìm.,

2000).

I-MCP is an ethylene antagonist that has recently generated interest for its use

on bananas. Golding et al. (1998), Jiang et al. (7999 a), Jiang et al. (1999b),

Harris et al. (2000), Jiang et al. Q000) and Macnish et al. (2000b) have

observed I-MCP effects on green bananas before ethylene initiation of ripening,

showing delayed ripening as measured by colour development, volatile

production and fruit softening.

Further I-MCP studies investigating extensions of shelf life (or eating life of

ripe fruit) have also been conducted on bananas that were ethylene-treated prior

to 1-MCP application (Macnish et a1.,1997; Golding et a|.,1999;Jianget al.,

1999b;Joyce et a1.,1999;Macnish et ø1.,2000a). These studies suggest that 1-

MCP applied to fruit after ethylene initiation of ripening extends banana shelf

life, but the studies have not provided sufficient evidence about the quality of

122

Chapter 8: Post-ripening: I -Methylcycloproper, e Exposure

fruit at the eating ripe stage (fully yellow) as perceived by consumers. As l-

MCP has not yet been approved for food products in Australia and other

markets such as the US, a relatively new sensory insfument, lnown as a

chemical nose, was used to identiff any sensory dif[erences due to I-MCP

application to bananas. Most of the previous reports also did not investigate the

application of I-MCP to ethylene initiated bananas after alengthy (>24hr)

delay after ripening initiation. Even though Jiang et al. (1999b) suggest a delay

in ripening is only possible when I-MCP is applied in the earliest phase of

banana rþening, research into 1-MCP application atlater ripening stages (>24

hrs after ethylene initiation) has only briefly been studied byMacnish et al'

(1991) and Joyce et al. (1999) Again these studies do not provide banana

quality data necessary for industy and consumers.

Desirable banana characteristics consist of a ripe product, with a shelf life (days

between ripening stage 4 to 7, CSIRO,1972) of greater than three days

(Ausüalian Banana Grower's Council, 1999). Therefore, the objective of this

studywas to examine the external and internal quallty effects of various I-MCP

concentrations and exposure periods applied 48 hrs after ethylene initiation.


8.2.1 Plant material and preparation

Green and hard banana (Cavendish cv. Williams) fruit were acquired during

January and February 2001 from a Tully property in North Queensland. They

were transported and prepared as outlined in sections 3.1 and3.2.

Twelve batranas were randomly assigned to each fieatment. Fruit were placed

into l0-L plastic containers in the presence of 160 g of Ca(OIÐ2 (a carbon

123

Chapter 8: Post-ripening: 1 -Methylcyclopropene Exposure

dioxide scrubber) and a saturated KNO3 solution that established at 95o/o relative

humidity. The containers were placed randomly in temperature-confolled

rooms at 20"C and banana ripening was initiated \Mith 300 pL Lr of ethylene gas

injected into the containers on two consecutive days. All containers were

ventilated for 30 min. each day, for the entirety of the experiment.

8.2.2 l-Methylcyclopropene exposure dfter ethylene initiation

After 48 hrs of ripening initiation, containers were ventilated and bananas were

treated with 0 (non-treated), 3 nL L r, 300 nL Lr or 30,000 nL Lr of I-MCP at

20"C. These I-MCP atmospheres were created from Ethylbloc@ 13.3% active

ingredient I-MCP, Rohm andHaas, Melbourne). Measured amounts of

Ethylbloc@ were placed into small plastic cups and placed into the lO-L

containers. Reverse osmosis (RO) water at 40'C in a ratio of I g Ethylbloc@ to

16 mL RO water (G. Baynon, Rohm and llaas, pers. coÍìm., 2000) was added to

Ethylbloc@ containers to release I-MCP. Al1 containers were sealed

immediatelyupon mixing and placed into 20"C controlled environments.

Fruit were exposed to 1-MCP atmospheresfor 24,48 or 72 hr. Containers were

ventilated daily and I-MCP reapplied as appropriate. At the end of the

treaünent period, bananas \¡/efe removed from containers, placed into unsealed

polyethylene bags, and rehrned to20"C for daily assessment until the end of the

experiment.

8.2.3 I-MCP detection

1-MCP concentation was quantified using a Varian gas chromatogaph model

3400 (Varian Ausfralia, Mulgrave, Victoria) fitted with a flame ionisation

detector and a stainless steel column (60 cm x3.ll5 mm id) packed with

124

Chapter 8: Post-ripening: l-Methylcyclopropene Exposure

80/100 mesh Porapak Q (Alltech). Column temperature was set at7Ùoc, while

the injector and detector temperatures were 135'C and 150oC, respectively.

Flowrates of air, hyd'rogen and the carrier gas nitrogen were 300,40 and 50 mL

ilh.-I, respectively, and the standard gas used to calculate I-MCP

concentrations was iso-butylene (103 pL U1; Oiang et al.,l999a).

8.2.4 Banana assessment

Six fruit from each treatment were used to measure external characteristics,

such as peel colour (green and shelf life), weight loss and discolouration, as

detailed in section 3.5. The remaining six bananas assigned to each treatnent

were used to assess internal quality parameters at colour stage 6; assessments

included pulp firmness (kPa), soluble solid concentrations (SSC o/Ð and aroma.

All internal qualitymethods were described in section 3.6.

8.2.5 Statistical analysis

The experiment was established as a 4 x 3 randomised complete block design

with three replicates. Four I-MCP concentations including 0 (non-treated), 3

oL Lt, 300 nI L 1 and 30,000 nL Lr were applied to bananasfor 24,48 or 72

hrs. Fruit in weekly replicates were randomly allocated to treatments. One

treaûnent unit for each replicate consisted of T2bananas, with six bananas used

for external quality assessments and the remaining six for internal quahty

assessments. Analysis of all data was performed using the General ANOVA

command in Genstat 5, (Release 41,4h edition, 1998, Lawes Agricultural

Trust, Rothamsted Experimental Station). Total weight loss data were

hansformed into reciprocd, data, firmness data were squared and all shelf life

data were logi¡ transformed for analysis purposes. Means, however, have been

back transformed for presentation purposes. A least signif,rcant difference test

),25

Chapter 8: Post-ripening: I -Methylcyclopropene Exposure

(LSD) atthe 5o/o level was used to examine all teatment effects. Chemical nose

results were analysed using the Pirouette multivariate dataanalysis package

(Infometrix Inc, Woodenville, WA USA), as described previousþ in section

3.6.3.1.

8.3 Results

I-MCP exposure periods did affect banana shelf lives with those exposed to 3

nL L:l I-MCP for 24 and 48 hrs having a single extra day of life, compared to

those exposed for 72hrs (Table 8.1). Shelf life judged as days to ripen from

ripening stage 4 to 7 of contol bananas was 3.3 days. 1-MCP at3 nLLt did not

increase this shelf life period, and 30,000 nL Lr I-MCP prevedted the ripening

of peel and flesh of most fruit and a shelf life could not be calculated.

Therefore the 30,000 nL L1 fieatment is absent from the following tables.

However, applications of 300 nL Lr 1-MCP doubled the shelf life of bananas to

an average ofgreater than six days.

t26

Chapter 8: Post-rþening: l-Methylcyclopropene Exposure

Table 8.1: Shelf life of Cavendish bananas cv. Williams following ripening at

20'C with 300 pI L I ethylene gas for 48 hrs then subsequently exposed to 0, 3

or 300 nL Lr I-MCP for 24,48 or 72 hrs at 20'C.

Exposure period ftns)

I -MCP concenúation (oL Lt) 48 7224

0

J

300

7,5.5 A

4.4 abc

7.3 d

3.6 ab

5.1 bc

7.2d

3.1 a

3.2 a

5.8 cd

"Dataare means of 3 replicates, where different superscripts denote significant

differences at P<0.05 using a LSD test. The data has been back transformed

from data that has been lo916 fansformed.

Green life and pulp firmness át colour stage 6 was similar for bananas treated

with 0, 3, or 300 tiL L-r I-MCP and for all exposure periods (Table 8.2 and 8.3).

Soluble solid readings át stage 6 averaged between 22-24% for all treated

bananas, except those exposed to 300 nL Ll I-MCP for 72hrs, which had lower

SSC values of an average of I 8.8% (Table 8.a). Weight loss readings for

bananas treated with 0, 3, or 300 nL Lr I-MCP were betwe enl.7-2.0%oand did

not impact on the visual presentation of the fruit (Table 8.5).

127


Tabte 8.2: Green life of Cavendish banan as cv . Williams following ripening at

20'C \¡/ith 300 pL L I ethylene gas for 48 hrs then subsequently exposed to 0, 3

or 300 nL L-l 1-MCP for 24,48 or 72 hrs at 20"C.

Exposure period (hrs)

I -MCP concenfration (nL Lr) 48 7224

0

3

300

4.0 f3.8 a

4.2 a

4.3 a

4.3 a

3.4 a

4.0 a

3.6 a

3.9 a

'Datzare means of 3 replicates, where similar superscripts denote non

significance at Þ0.05 using a LSD test.

Table 8.3: Pulp finnness of Cavendish bananas cv. Williams at ripening stage 6

following ripening at 20'C with 300 pL Lr ethylene gas for 48 hrs then

subse{uentþ exþosed to 0, 3 or 300 nL L1 I-MCP for 24,48 or 72 hrs at 20'C.


I -MCP concentration (oL L-t) 48 7224

0

aJ

tt4 f143 a

125 a

109 a

Ill a

146 a

126 a

140 a

ll2 a300

" Dataare means of 3 replicates, where similar superscripts denote non

significance at P>0.05 using a LSD test. These data have been back

transformed from squared data.

128

Chaper 8: Post-ripentng: l-Methylcyclopropene E¡posure

Table 8.4: Soluble solid concentation of Cavendish bananas cv. Williams at

ripening stage 6 following ripening at20"C with 300 pL Ll ethylene gas for 48

hrs then subsequently exposed to 0, 3 or 300 nL L-r I-MCP for 24,48 or 72lvs

at20"C.

Expostre period (hrs)

I -MCP concentration (oL L-t) 48 7224

0

a--)

23.3 f22.1a

23.7 a

23.1a

23.0 a

22.7 a

23.0 a

22.2 a

18.8 b300

'Dataare means of 3 replicates, where different superscripts denote significant

differences at P<0.05 using a LSD test.

Table 8.5: Weight loss (7o) of Cavendish bananas cv. Williams from ripening

stage I to 6 followingripening at2}"Cwith 300 þLLr ethylene gas for 48 hrs

then subsequentþ exposed to 0, 3 or 300 nL Ll I-MCP for 24,48 ot 72 hrs at

20"c.


1 -MCP concenûation (nL Lt) 48 7224

r.7 * 1.7 a 1.8 ab

1.7 a 1.8 ab 1.8 ab

300 2.0 c 2.0 c 1.9 bc

'Datzare means of 3 replicates, where different superscripts denote significant

differences at P<0.05 using a LSD test. This data has been back transformed

from reciprocal data analysis.

0

J

129

Chapter 8: Post-rþening: l-Methylcyclopropene Exposure

Bananas exposed to 30,000 nL Lr 1-MCP showed significant quality

differences compared to all other treaûnents, because they had to be examined

at ripening stage 2 þeel was green with a fface of yellow, Figure 8.1) as fruit did

not ripen before fungal spoilage (crown rot) ocourred. Firmness and SSC were

596 kPa andSo/o,respectively, but weight loss (2%) was not dissimilar to those

treated with 0, 3 nL I;1, or 300 nL Lr I-MCP. Shelf life and aroma could not be

measured, as fruit did not ripen.

130

Chapter 8: Post-rþening: l-MethylcyclopropøT e Exposwe

Figure 8.1: Cavendish banana (cv. Williams) appearance on day 8 of the l-

MCP experiment. Bananas were initiated with 300 FL Lt ethylene gas for 48

hrs at 20'C and then exposed to 1-MCP for (A) 0 (air), (B) 3 nL Lt, (C ) 300 nL

Lt or @) 30,000 nLLl.

131

Chapter 8: Post-ripening: l-Methylcycloproper, e Exposwe

Banana aromas of all treatments that could be analysed at ripening stage 6 were

similar (Figure 8.2). The PCAmodel identified 86% of the total aroma variance

between treatnents to consist of two factors; 7 5%io of the variance was related to

Factor I andlL%oto Factor 2. My remaining factors each made ap < 5o/o of the

total variance. Variability attributable to treatments was limited when

compared to non-treaûnent factors, as expressed by the similar dispersal of

values around Factor 7 and2 for all fieatments shown (Figure 8.2). Therefore

when observingthe loadingplot (F go.e 8.3), the ions (61, 71,70 and 57 or

variables) present at the exüeme ends of the factors are similar in all samples.

Aroma compounds were identified with the aid of an established volatile

compound library used in conjunction with the chemical nose loading plot

(Figure 8.3). Volatiles identified from the banana samples were butanoic acid,

butyl ester, l-butanol, 3-methylbutyl esters and ethyl acetates, giving bananas a

fruityripe aroma with a slight over-ripe note.

132

Chapter 8: Post-rþening: l-Møhylcyclopropene E4osure

6A

f^

A

A

(\ãlg

-10

Fadol

Figure 8.2: The chemical nose score plot of volatiles produced by Cavendish

bananas cv. Williams held for 24,48 or 72hrs in 3 nLL-1 I-MCP (4,- ),

300 nL Lr I-MCP (8, -

) or in air (C,- ) after ripening initiation.

133

Chapter 8: Post-ripenurg: l-À¿tetloylcyclopropene Exposure

ôtoo6L

Factorl

Figure 8.3: The loadings plot of ions (væiables) fot ripe Cavendish bananas ov

Williams exposed to 3 nL L1 I-MCP, 300 nL Lt I-MCP or held in air after

ripening initiation for 24,48 or 72 hrs in.

t34

Chapter 8: Post-ripening: I -Methylcyclopropene Exposure

8.4 Discussion

The process of ripening and senescence of banarias is influenced by their

production of endogenous ethylene. I-MCP stops ethylene action, including the

promotion of fi.uther ethylene production, by binding to the ethylene receptors

(Sisler and Serek, 1997). As mentioned in section 8.2.2,a1lbananas were

ripening initiated with ethylene before 1-MCP application. Therefore,

endogenous ethylene production and the ripening process was in progress when

I-MCP was applied.

Only in bananas treated with 300 nL Ll I-MCP was ethylene action slowed to

gtrve acommercial extension at shelf life. The ripening of peel and flesh of all

fruit was qmchronous as seen from the typical and concurrent ripening changes

observed in the peel and flesh. This is encouraging, because previous research

by Jiang et al. (1999a) had reported uneven peel degreening in batranas treated

with I-MCP prior to ripening. Uneven degreening or ripening of fruit is

undesirable and would be unacceptable to industry. Ripening uniformity may

have been aided in our experiment by elevating bananas in treafnent chambers

to allow all-over airflow and therefore chemical absorption to all sides of the

bananas; this would be established commercially using fan-forced airflow in

ripening chambers.

Low concentrations of I-MCP (3 nL Lr) applied after ethylene initiation were

not successful in extending shelf life, possibly as ethylene was able to

successfully compete with I-MCP for ethylene binding sites as reported by

Sisler and Serek (1997). Jianget al. (1999b),however, showed that ethylene

inhibition by I-MCP was non-competitive by measuring the affinity of I-MCP

and ethylene to binding sites, and observing that I-MCP had a lower binding

135


constant (Ç) than ethylene. Therefore, I -MCP has the grcater athaction for

ethylene binding sites. However, there may have been more binding sites than

l-MCP molecules available to bind to them, or I-MCP did not persist in the

tissues to block the continuously forming ethylene receptors (Jiang et al.,

1999b). This consequently allowed ethylene to continue to promote ripening.

The ethylene induced ripening process of bananas treated with 30,000 nL Lt 1-

MCP was delayed, with many fruit failing to ripen before crown rot infection

detracted from their appearance. Failure of fruit treated with a high I-MCP

concentration (30,000 oL Lt) to ripen, can be explained by the limited

fonnation of new ethylene receptor binding sites (Jiang et al.,1999b), the full

occupation of existing binding sites or persistence of 1-MCP in tissues. During

the period of ethylene insensitivity and inhibited ripening, infection of fruit by

fungi is not uncommon (Sisler and Serek, 1997),as with the crown rot

infectiohs observed on 30,000 nL Ll 1-MCP treated fruit during the prolonged

storage period. Fruit infected with crowlr rot continue to deteriorate as ripening

advances (Jones et al.,1993), reducing banana saleability.

The chemical nose was used because it is a quick, easy and reliable instrument

that produces comparable results to time consuning sensory panel findings

(Klieber and Muchui,2002). Also the use of I-MCP on food products in

Australia has yet to be approved, so taste panels could not be used. The similar

score plots for all ripe fruit suggest that bananas that were or were not I-MCP

treated all had similar aroma profiles. It is therefore unlikely that consumers

would be able to detect aroma differences from I-MCP treatedfruit.

The momas identified in the analyses were butanoic acid, butyl ester, l-butanol,

3-methylbutyl ester and ethyl acetate, which exhibit the bananalike and fruity

136

Chapter 8: Post-rþening: I -Methylcyclopropene Exposure

aromas (Tressl and Jennings,l972;Marriott, 1980) that are expected from ripe

bananas. Chemical nose results presented in section 8.3 showed that the

treatonents of 1300 nL I;l I-MCP on ethylene initiated bananas for various

exposure periods had no apparent qualitative effects on the composition of

banana aroma profiles when compared to normallyripened bananas and so

similar banana aromas would be perceived by consumers, although Golding er

al. (1999) reported significantly reduced quantitative production rates in

volatiles that evolve in ripeningbananas using GC-FID after I-MCP ûeaünent.

8.5 Conclusion

l-Methycyclopropene is a pfotTrising postharuest üeatrnent for the extension of

banana shelf life to more than 6 days at 20oC when apþlied dfter ripening

initiation. Qality of banana flesh as well as visual appeatance was not affected

by 1-MCP treafnents of <300 nL L-l. However, the I-MCP concenftation used

is critical, as low cohcerittations (<3 nL Lt) did not extend banána shelf life and

high cohcentrations (30,000 oL Lt) caused fruit to be u-râbceþtable for

consurnption. I-MCP is not yet approved for use on fruit and vegetables in

Australia, but is approved on food products in Chile and on cut flowers in the

USA.

137

ëhàpter 9

9. General Discussion

9.1 Bacþround into iildustry re{uifements

Asian and in particular the Philippine's interest in establishing a banana export

market to Australia has become apparent in recent years. This has increased the

need for research into local banana production and management, focussing on

consumer and market demands such as extending banana shelf life and

maintaining a yellow peel appearance on ripe fruit all year round. Cavendish

cv. Williams barìanas were used throughout this research because they account

for 95o/o of banana production in Aushalia @aniells, 1986).

This study was ai¡red at improving the quality and overall posthafvest life and

in párticular the shelf life of bananas. This included looking at suitable

conrmercial methods for the eady detection of chilling injury, a physiological

disorder that decreases the appearance and the value ofbananas. This would

allowmarkets to redisü'ibute chilled and discoloured fruit to appropriately

graded markets and prevent the sale of lower quality fruit to premium markets.

Investigation into current commercial ripening procedures was also required, as

the quality of bananas varies throughout the year, so the posthawest

management of ripening temperaflnes and ethylene concenfration throughout

the year should be considered as possible influential factors in seasonal banana

138

General Disct¡ssion

quality. Furthermore, changes to existing ripening procedures such as exposure

periods to nitrogen, ethanol and l-methylcyclopropene afinospheres were also

investigated to extend banana shelf life.

9.2 Pre-ripening detection of chilling injury

Chilling injury is a physiological disorder that causes browning of the vascular

tissue and is clearly visible on ripe bananas (Marriott, 1980; Stover and

Simmonds, 1987, Kays, 1991; Wang and Gemma,I994). It is of importance to

the banana industry, because consumers relate the general appearance of the

peel with overall quahty(Kays, 1999). Mild chilling symptoms onlybecome

apparent after ripening, which causes problems for the industry. Early detection

of chilling injury would allow industry to identifu chilled fruit and market them

by grading them and allowing quality fruit to reach premium priced mækets.

This study was based on finding and testing a suitable method that could detect

chilling injury on bananas. It needed to be relatively quick, easy and suitable for

industry use. Chlorophyll fluorescence was targeted, as a result of findings by

DeEll et al. (1999); they described chlorophyll fluorescence as an easy method

of measuring processes that take place in chloroplasts. Smillie (1979) had

previously reported chilling injury to cause disruption to chloroplasts.

Therefore, theoretically, chlorophyll fluorescence measurements could detect

changes to the photosyrthetic ability of chloroplasts from any disruption due to

chilling injury. PPO activity in bananas after chilling was also investigated.

En4mratic oxidation byPPOs is the cause of the brown pigment formed in

vascular tissues of chilled fruit (Palmer,1963 Jones ef a1.,1978). As PPOs are

bound to chloroplasts especially on the inner surface of the thylakoid membrane

(Shomer et a1.,1979;Amiot et a1.,1997) and chloroplasts are disrupted by

139

General Discussion

chilling the activity of PPO in chilled fruit could be different to that of non-

chilled fruit. However, Gooding et al. Q00l) found almost no change in PPO

expression during ripening even after induced mechanical injury and found a

relatively constant PPO activitythroughout all ripening stages.

No differences were observed in chlorophyll fluorescence between chilled and

non-chilled fruit, as the measurement of chlorophyll fluorescence was on

epidermal chlorophyll not the sub-epidermal tissue where chloroplast

breakdown led to browning. PPO activity was also found to be unsuitable for

the early detection of chilling iojury, because bananas did not show a wound

defence response in the form of an increase in PPO activity after chilling.

Therefore, a method for the early detection of chilling injury was not found.

9.3 Optimisltg ripening tempèfâtüles dild ethylene concentrations

throughout the year

Optimising ripening temperatures and ethylene concentrations used by

commercial enterprises throughout the year was investigated. Ripening

temperatures used commercially vary between 14 and 20"C,with market

demand for faster or slower ripening rather than banana quality being the

temperature detennining factor. Industry also currently uses 300 FL Ll of

ethylene to initiate ripening but no evidence has been found why this exact

concentration is used (8. Charfes, Chiquita Brands South Pacific, pers. comm.,

leee).

Seasonal effects on banana ripening quality have briefly been studied by Young

et al. (1932) and Rippon and Trochoulias (1976),but a detailed study had not

been undertaken. This is the main reason ripening procedures were investigated

740

General Discussion

throughout the year. The aim of this studywas to observe the ripening

treaÍnent effects on the intemal and external quality of bananas throughout the

year.

This study found the lower temperatures of 14 and l6'C slowed the rate of

respiration (Couey, 1982; Wills et aL.,1998), which extendedbanana shelf life

to 9 days, but produced symptoms of mild and severe peel discolouration in the

July and September harvests. Bananas ripened with the higher ripening

temperatures of l8 and 20'C had the appearance of slight peel discolouration

only after a winter growth period and generallyhad sheHlives of between 4-6

days. Impoftantly, it also showed the effect seasonal development had on

postharvest quahty, with bananas that grew and developed during winter

affected by in-field chilling which becarne apparent after ripening. The severity

of the in-f,eld winter chilling was álso found to be affected by postharvest

ripening procedures, with lower ripening temperatures increasing the severity of

peel discolouration. Ethylene concefltratiohs between 50 and 1000 pL Ll used

to itliti¿te ripening did not affect ripeititlg plocesses or banana quality, so the use

of less or more than 300 pL Lr of ethylene in industry through ripening room

leakages or accidental additional gases, respectively, would not be defimental

to the quality of ripened bananas.

This research identifies the need for commercial enterprises to monitor in-field

temperatures throughout banana growth and development, to ensure visually

acceptable bananas. Monitoring is especially essential for those in the banana

indusfythat continue ripening fruit upon market demand, as this is when lower

ripening temperatwes are used to manipulate the ripening process. The use of

lower ripening temperatures in this study was fourd to influence the degree of

t4t

General Discussion

discolouration found on bananas, especially during winter developmental

periods and sudden climatical events such as cyclones.

9.4 Post-climacteric extension of shelf life

9.4.1 Nitrogen atmospheres

Nitrogen exposure after ethylene initiation was investigated in this study to

extend research by Wills et al. (1990) who used this approach to extend green

life of bananas. Their research found stage I bananas exposed to nitrogen

storage for 3 days showed a42%o increase in green life. Furtherrnore a study by

Parsons et al. (1964) detected onlynegative affects on aroma if bananas were

stored under nifrogen for longer than 4 days. Therefore, nitrogen atmospheres

were used in this study to displace oxygen and in the process to reduce

respiration rates, ethylene s5mthesis and ethylene receptor affinity after ripening

initiation to extend banana shelf life rather than green life.

Unfortunately, nitrogen exposure after ethylehe initiation cannot be

recommended based on this research. Exposure to nitogen atmospheres after

48 hrs of ripening initiation did not extend banana shelf life, but exposure

periods of as little as 6 hours caused discolouration of banana peels, which is

unacceptable in the markeþlace.

A further experiment established that low oxygen injury was the cause of the

peel discolouration observed and not low humidity. Research by

Akkaravessapong et al. (1992) had foturd that lowhumidity caused

mechanically damaged tissues to dry causing black discolouration. However,

the work on humidity effect in combination with nitrogen exposure confirmed

initial conclusions that peel discolouration was due to the low oxygen

142

General Discussion

atmosphere (Wills et a\.,1990; Thompson, 1998).

9.4.2 Ethanol atñospheres

Ethanol is a product of anaerobic respiration, which can be indirectly increased

via modified or controlled atmospheres @esis and Avissar,1989; Pesis and

Marinansþ , 1993; Pesis, I 994; Pesis , 1996). Studies have focussed on better

control over the process by directþ applytng ethanol to tomatoes (Salreit,

1989; Saltveit and Sharaf, I992;Hong et a1.,1995; Beaulieu and S¿ltveit,1997),

bananas and otherproduce (llewage et aL.,L995;Ritenour et aL.,1997).

However, ripening inhibition was not found in bananas, unlike in tomatoes

where ethanol was absorbedthrough the stem scar or fruit peel. In both banana

experiments, fruit were heid in atrnospheres created by the application of

ethanol to filter pdper. However, no measurements of ethanol concentrations in

banana peel or pulp were conducted, so the absorption of ethartol into the

bdnanas was not clear.

A recéht improvement in the application of ethanol to tomatoes was repofted

by Ratanachinakorn et al. (1999), using vacuurn infiltation. This method is a

rapid and forceful way of increasingthe volatile content of fruit tissues.

However, unlike irr the tomato study, ethanol was not absorbed into the pulp of

bananas and consequently ripening was unaffected. This suggests that further

research into forcing exogenously applied ethanol atnospheres into thick

skinned produce is needed, but also encourages the use ofindirect approaches

such as modified and conüolled atrnospheres to increase internal ethanol

concentrations in bananas, as Pesis and Marinansky (1993), Pesis (1996) and

Ratanachinakorn et al. (1997) did with tomatoes.

143

General Discussion

9.4.3 l-Methylcyclopropene atmosphefes

The quality of bananas a.fter exposure to I-MCP, an ethylene antagonist, that

irreversible binds to ethylene receptors and prevents the production of

endogenous ethylene, effectively preventing ripening (Sisler and Serek, 1997),

was examined.

Use of I-MCP on ethylene initiated bananas, as in this study, has been

investigated before byMacnish et al. (1997), Golding et al. (1999),Jianget al.

(1999b), Joyce et al. (1999) and Macnish et al. Q000a). However, even though

the studies did provide infomration on banana shelf life and in one case on

aroma volatile production, the research did not provide infonnation on the

quality of bananas in relation to consumer preferences.

Investigation into the lengttr of I-MCP exposure was also conducted in this

study, as Jiang et al. (1999) reported the availability of new ethylene binding

sites for the recurrence of ethylene synthesis after I-MCP treaÍnent. The

extended periods of l-MCP exposure after initial ethylene contact was aimed at

delaying ripening for as long as possible

In this study, bananas held in 300 nL Lt I-MCP after ethylene initiation were

found to double in shelf life without negatively affecting quality. However, the

1-MCP concentation applied is critical, as the lower (3 nL Ll) and higher

(30,000 oL I;t) concenfations used were not successñrl in extending shelf life

or totally inhibited fruit ripening and were susceptible to crown rot infection,

respectively. A more detailed analysis into the concentrations between 3 nI- L-1

and 30,000 nLLl wouldbe usefrrl, as further shelf life extension maybe

possible.

t44

General Discussion

The longer I-MCP exposure periods Q-2a hrs) investigated in this studywas

examined to observe whether the longer exposure periods also increased the

shelf life of bananas. Extended exposrre did not affect the efficacy of the

treatrnent, wifh24 hr treaÍnents equally as effective as72hr teahnents.

However, researchers have yet to determine a standard length of exposure

suitable for commercial enterprises with reported holding times and I-MCP

concentrations varying from 1 to 24 hrs and 300 to 45,000 nL L1, respectively.

Jiang et al. (1999b) found the effectiveness of I-MCP was dependent on the

length of I-MCP exposure and concenhation, with higher concentrations

required if short exposure periods were used. Before commercial markets can

use this ethylene antagonist, a study into a standardised application progranìme

for industry is essential, preferably using a high I-MCP concenhation for a short

exposure period to allow the treatnent of large quantities of produce in the

shortest time possible. However, quality of fruit must be monitored if the use of

higher I-MCP concenfiations is to be used, as uneven peel degreening was

observed on bananas after24 hr exposure to concenfrations of 500 and 1,000 nL

Lr of I-MCP (Jiang et al.,I999a). Uneven degreening in the previous study

may have been influenced by the lack of 1-MCP diffirsion into peel by

inadvertentlyblocking surface pores @erez and Beaudry, 1998). Even

degreening was promoted in this study by raising the fruit, thus allowing I-MCP

exposure to the entire banana surface.

Further investigations into using I-MCP throughout the year need to be

conducted to determine whether bananas grown during difÊerent seasons react

differently to I-MCP exposure, as their ripening behaviours varied at different

times of the year in this study. Also lower temperatures have been shown to

reduce the effectiveness of 1-MCP binding of ethylene receptors (Sisler e/ a/.,

t45

General Discussion

1996; Sisler and Serek, 1997) andtherefore winter grown produce may react

differently to I-MCP exposure compared to summer grown produce.

9.5 Commercial benefits from this research

This study has allowed a better understanding of ripening procedures in relation

to shelf life, visual appearance and internal quahty of bananas, when grown

under various climatic conditions. Currently industry ripens bananas at

temperatures between 14 and2O"C with 300 pL L1 of ethylene apptied for the

first two days of ripening, but ripening temperature depends entirely on market

demand. Higher temperatures are used to speed up rþening and lower ones to

delay ripening. Findings have identified the importance of recording in-field

climatic data and events to assist in deciding the optimal ripening conditions of

bananas. A ripening procedute suitable for any time of the yem would consist

of ripening mâture, hard, green, stage I barranas at 18'C with between 50-300

pL Lr ethylene on two consecutive days, resulting in a banana shelf life of

approximately 6 days. Ripening bananas at20"C would also result in a good

visual appearance, but would reduce shelf life (a dayÐ in some months of the

year. If monitoring of field temperatures is conducted throughout the growth

and development period and temperatures are fotrrd to be above l3"C without

any occuffence of sudden climactic changes, such as cyclones, ripening

temperatures of 14 or 16oC would increase shelf life of bananas without

increasing peel discolouration. The use of between 50-300 gL Lt of ethylene to

ripen bananas will avoid excessive ethylene use and associated costs, with

similar banana quality resulting from the lower concentrations.

Apromising post-climacteric treatment for extending banana shelf life of

ethylene-initiated bananas was exposure to 300 nL Lr of l-methylcyclopropene.

146

General Discussion

This level significantly improved banana shelf life without affecting peel or

pulp quality. I-MCP is approved on food products in Chile and approved on cut

flowers in the USA. It is yet to be authorised on food products in Australia, but

will prove veryusefrrl if its approval is granted, which is likelyto occur in the

near future.

9.6 Future research

Even though this research has allowed an insight into the physiology of

bananas, fuither research into the following areas is required.

Continued research into an early detection of chilling injury is required. This

remains the prirnary cause for peel discolouration and while we now know the

severity of the discolouration is affected by postharvest managemettt, the early

detection of chilled fruit would allow management of those affected to be

ripened separátely from those not affected by in-field chilling. Investigation

into the use of the chetnical nose in association with the early detection of

chillitrg injury in bananas would be of benefit to wholesalers. Banana peels at

the green, mature stage, may indicate differences in aroma profiles, as the

presence of browning substances may cause a change in peel arolna (Mabrouk,

1979). Earlier research by Murata (1969),found higher acetaldehyde and

ethanol concentrations in severely chilled green banana peels compared to

healttry green banana peels, which possibly influenced their aroma profiles.

Furttrer analysis into the early detection of peel discolouration may focus on the

differences observed bywholesalers between bananas growTt in low compared

to high sunlight hours (T. Johnson, Chiquita Brands South Pacific, pers. comm.,

2001). Seasonal variation in PPO activity of bananas may be a method for early

147

General Discrssion

peel discolouration detection. The effect oflfV or sunlight hours during

seasonal growth may influence banana PPO activity, because gamma irradiation

on bananas has been found to positively correlate with PPO activity and skin

discolouration (Thomas and Nair, l97l). This may in-tum lead to further study

into the types of bunch covers used by industry to protect bananas from peel

discolouration due to changes in sunlight hours during seasons.

Field applications of bunch covers (different materials or thicknesses at

different times of the years) require some investigation. Light peneüation is

especially important dtring flìe months of January and February when low

sunlight hours affect peel coloutation (T. Johnson, Chiquita Brands South

Pacific, pers. cornm .,2001),reducing peel appearance. This could lead to

various bunch covefs being trialled throughout the year depending on weather

patterns.

Future investigation into the extension of banana shelf life needs to focus on the

indirect increase of ethanol in the early stages (1-4) of ripening using modified

ahnospheres. However, continual study into developing an exogenously applied

technique which is able to force ethanol into the pulp of bananas would be

beneficial; as it would allow commercial markets confol over the ripening

process.

Additional research must also be conducted into the application of I-MCP in a

commercial environment, ensuring total banana exposure to I-MCP preventing

the uneven degreening that has been previously observed. This research is

essential during the time before its approval on foods. During this time,

investigation should also include the use of different levels of I-MCP

148

General Discussion

throughout the year, to ensure its effectiveness all year round.

All of these areas will provide the banana industry with extra data that will

ultimately lead to better management practices in the field as well as

postharvest. Únproved management will ensure bananas with the best

appeârance, taste and aroma are distfibuted to consumers worldwide. It will

also encourage Aushalian consumers to buyAusfralian produced quality

bananas, rather than bananas which may become available through Philippine

imports iil the near futtlre.

149

,Biblíogiepbü

(a) Puhlications from this thesls (reprints located at back)

Jorlrhal artlcles

Klieber, 4., Bagnato, N., Barrett, R. and Sedgley, M. (2002). Effect of post-

ripening nitrogen aÍnosphere storage on banana shelf life, visual appearance

and aroma. Postharvest Biologt and Technolog,t,25: 15-24.

Bagnato, N., Klieber,4., Barrett, R. and Sedgley, M. (2002). Optimising

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Bagnato, N., Klieber,4., Barfett, R. and Sedgley, M. (2002). Effect of ethanol

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Conference abstracts

Bagnato, N., Muchui, M., Klieber, 4., Barrett, R. and Sedgley, M. (2001).

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Australasian Postharvest Conference ,23-23t' September, Adelaide, Ausüalia.

Kliebeq 4., Bagnato, N., Barrett, R. and Sedgley, M. (2001). Effect of post-

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Klieber, A., Bagnato, N., Sedgley, M. and Barrett, R. (2002). Banana Ripening:

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August, Toforlto, Canada.

Miscellnnbbús

Bagnato, N., Klieber,4., Bâffett, R. äiid Sëdgley, M. (2001). Optiñising

Cavendish riþetring procedtlres year round. Good Fruit and Vegetables, l2z 29

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NOTE:



ELSEVIER Posllrarvest Biotogy and Technology 25 (2002) I5-24

PostharvestBioloqv andTechñólogy

lvlvw.elsevie r.com/loca te/posttrar.v bio

Effect of post-ripening nitrogen atmosphere storage onbanana shelf life, visual appeatance and aroma

A. Klieber *, N. Bagnato, R. Barrett, M. Sedgley

Depffttnent of Horticutture' t/iticutture '"'' oä:,:"Ij:,:,,::;.::{'; i;;i,"';':,:,,:,:f;:""

ctunpLts' Ptcmt Research centre' PMB I'

Received 2l February 2001; accepted 2l August 2001

Abstract

Bananas (ù[Lrsct ctcturrinata Colla, Cavendish cv. 'Vy'iiliams') were stored in nitrogen at 22 oC lor 6, 12 and 24 h ata more gleen than yellow (stage 3) or: more yellow than gr:een (stage 4) r:ipening stage. Shelf life in nitroge¡ at22."C,that is the time taken lrom a more yellow than gleen colour stage 4 to yellow with slight brorvn flecking stage 7, rvas

not extended when compared to air-stored bananas. However, areas of brown discolouration appealed oLr banauasplaced in nitlogen-storage. The aroma of ripe bananas was assessed ',vith a mass spectrometly-based chemical nose.

Bananas stored in nitrogen genelally had a riper aroma prof,le compared with air-stolage. An ion with a mass tocharge ratio of 6I was strongly associated with nitrogen-treated bananas; this ion is a decomposition prodLrct of a

known banana aroma colnpound, ethyl acetate that produces an over-ripe banana note. An ion witl-r a mass to chat'ge

latio ol55 was associated with air-stored bananas; this ion is a decomposition product of ripe bananas (3-methylbutylester and 1-butanol). Post-climacteric nitrogen storage is not a suitable method fol incleasing shelf life, as it causes

skin browning. @ 2002 Elsevier Scieuce B.V. All rights reserved.

Keyworcls: Nitrogen storage; Btrnanas; Shelf life; Chemical nose; Aroma

1. Introduction

A consumer product survey prepared for theAustralian Banana Grower's Cor.rncil (1999)for-rnd that if bananas had a longer shelf life thanthe current 3 days, Ar"rstralians would be likely topurchase more of these fruit. CLrrrently, Aus-

* Corresponding author. Tel.: * 6l-8-8303-6651; fax: + 6L-8-8303-71 16.

E-ttail address: [email protected] (4.Klieber).

0925-521410215 - see lront rnatter O 2002 Elsevier Science B.V. All rights leserved

PII: S0925-52 t 4(0 t )00 I 63-6

tralians consrrme over l5 kg olbarranas per capitaper allnum (Australian Battana Grower's Council,1999). The short shelf lile is due lo a rapidsenescence process lhat causes the visual appear-

ances of the fruit peel to clegrade from yellow to a'muddy' brown colour telminating banana shelflife. Wholesalet's waut to extend the banana shelflife between ripening stage 4 (more yellor.v thangreen) and 7 (yellow with light brown flecks)(CSIRO, 1972) to inc¡ease appeal to consnmers togain higher purchase quantities at one shoppingoccaslon.

t6

Currently, ripened bananas are pre-cooled tol3 iC before their clistribution to supermarkets(T. Johnson, Chiquita Brands South Pacific,pers. commun., 2000), to slow down fruitmetabolism and therefore prolong senescence(Rippon ancl Trochoulias, 1976). However, this is

costly, and fruit rewarm rapidly on the displayshelves, thereby reducing shelf lifle.

Most research has focused on ways to extendthe gleeu lile of unripe lrr,rit. Modified atmo-sphere packaging has been suggested as a substi-tute for low temperature storage (Scott et al.,1970); however, this storage is costly as it in-volves labour as well as ethylene absorbent pack-aging and also requires carefr.rl handling toprevent damage to bags and loss of the modifiedatmosphere. Wills et al. (1990) successfully usedshort-term nitrogen treatments to increase thegreen life (fi:om harvest to yellow with green tips)of bananas, with nitrogen atmospheres of 3-dayduratiou applied immediately alter harvest in-creasing their green life by 42%. Nitrogen atmo-spheres are inexpensive and easy to apply tolarge qr.rantities ol fi'uit. A nitrogen atmospherewill reduce the level of oxygen available to thefruit, which in ttun will inhibit ethylene produc-tion and ripening (Thompson, 1998):

A method is required for slowing the rate olsenescence of ripening bananas to extend theirshelf life from' the point of sale to consurnptiou.The aim of this study was to examine whetherstoring fruit temporarily in nitrogen atmospl-reresafter partial ripening conld extend the shelf lifeof bananas wilhor"rt aflecting their appearance oraIoma.

2. Materials and methods

2.1. Pktnt materictl ancl prepa.ration

Chiquita Brands South Pacific Ltd. Pty. sr.rp-plied Cavendish bananas cv. eWilliams' used forthis study. Fruit were harvested during themonths of June and July 2000 in the Cairnsregion of north Queensland and transported at13 'C to the Plant Reseatch Centre (Waite Carn-pus, Adelaide University) within 3 days of har-

A. Klieber ct ttl. / Postlruruest Biolog¡, tutcl Tecltnology 25 (2002) l5-24

vest. All hands were harvested from plantswithin a single block. Only hands from the mid-dle of the bunch were used, to ensure an evenmatLrrity of frr.rit. At arrival a[ h.uit were stillgleen and hard. Once delivered, the bananaswere dehanded ancl dipped lor I min in hrngicide(0.55 ml/l Sportak, Hoechst Schering AgrEvo,Gler-r lris, Vic.) and allowed to air dry. Matchedfingers were then landomly allocated to tl.eat-ment groups. A treatment unit consisted ol 12flrtLit, six of which were nsed to assess externalcharacteristics of the fruit and the remaining sixwere nsed to assess internal properties of thebananas.

Bananas were ripened in l0-1 plastic containersin the pr'esence of a CO, scavenger (160 g ofCa(OH)r) ensr-rring a \Yo CO2 level, and a 95o/"relative humidity was maintained r-rsing a satu-rated KNO. solution. All fruit were ripened ac-cording to common industry practice to r-ipeningstage 3 (more green than yellow) or 4 (moreyellow than gleen) at 16 'C with 300 pl/l erhyl-ene gas on the fir'st two consecutive days ofripening (T. Johnson, Chiquita Blands Sor-rthPacif,c, pers. çommr-rn., 2000).

2.2. Nitrogen application for uarious periods oftitne at different ripening stages

Alter initiation of ripening, bananas werestored in a nitrogen atmosphere at ripening stage3 or 4. Storage peliods were 0, 6, 12 or 24 h.The two control (no nitrogen) treatments werestored in vented polyetl-rylene bags at 22 "C oncethe appropriate ripening stage had been reachedat 16 'C. Treatcd bananas were placed in con-tainers that were flushed with high purity nitro.gen (BOC gases, Adelaide) for 5 min, belolebeing sealed for the allotted tteatment periods of6, 12 or 24 h. Atmosphere samples were takenftom sealed containers and anaiysed using a thel-mal conductivity detector gas chromatograph(Varian 3300, Walnut Creek, CA) to ensure O,and CO, levels were < I and 0%0, respectively(Wills et al., 1990). On completion of treatments,lruit were placed in vented polyethylene bags at22 "C to complete ripening under simulated retailmarketing conditions.

2.3. Nin'oget1 ettllosphere sl.oroge in the presenceof low or hÌgh relatiue ltumidity

A second nitlogeu atmosphere expeliment wasperformed to observe whether humidity stresswas a factor in the results obtained fi-om theprevions experiment. Bananas were ripened tostage 4 as above and held in nitrogen lor 0 or 24h withor-rt humidity control. In addition, a simi-lar lot of bananas was held in nitrogen for 24 \tin the presence of a satul'ated KNO, solution(95% RH). The atmospheric composition wasagain monitored. After nìtrogen treatment, ba-r.ìanas were removed from stolage containel's andplaced in polyethylene bags. To regulate humid-ity, fruit were held in the presence or- absence ofa beaker of water in the unsealed bags. Air-stored fruit were stored in closed bags that werefolded under the bananas without a beaker ofwater. The relative humidity inside polyethylenebags was tleasured using a hand-held humidityprobe (HM34C, Vaisala, Helsinki) as > 95% RHfor high (beakers of water present) or <75o/,RH for low humidity treatnrellts.

2.4. Assessntents of bqncuzcts

2.4:. 1. Extentttl assessmenÍsAll bananas were weighed at ripening stage I

and at ripening stage 6 to determine the percentweight loss. In addition, silelf life was determinedby observing banana peel colour changes as de-termined by the CSIRO Banana Ripening Chart(CSIRO, 1972) and calculating the number ofdays ìt took for the peel to change lrom ripeningstage 4 (more yellow than green) to ripeningstage 7 (yellow with light brown flecks), exclnd-ing tleatment periods.

Discolouration of the banana peel was ob-served at lipening stage 6 (fully yellow). Thediscolouration severity scale used consisted of 0,1ro discolouration; 1, slightly discoloured; 2,mildly discolouled; 3, severely discoloured. Asfi-uit from a given treatmerìt displayed similarsymptoms, scores were weighted and averagedinto ar.r O, injury discolouration index (DI) foreaclr treatment, using a fol'mula modified fromSchin'a et al. (1999):

A. Klieber et ol. fPosrharuest Biologv mù Tethnolog¡, 25 (2002) l5-24

(a x 0) * (ó x l) + (c x 2) -r (d x 3)(t)

t'7

11

where r¡ is the number of fi'r.rit with no dis-color.rration, å the nurlber ol fi'uit with slightdiscolouration, c the number of fruit with ruilddiscolouration, r/ the uullber of fruit with sevel'ediscolouration, and ¡l the total nullber of fruitobserved.

2.4.2. Intental fruit assessm etxts

Pulp firmness is a measure of the crushingstr-ength of pulp tissue aud v/as n-ìeasured using atwist tester (Depaltrnent of Agricr-rltural Engi-neering Massey University, NZ) as desclibed byHarker et al. (1996). This method is destructiveto fi-uit tissue; however', it is very easy to use andgives reliable ¡esults wheu used on soft tissue,unlike a hand-held penett'ometer (Studman andYuwana, 1992). The penetrometer's action con-presses the flesh against flesh within the fruit,whereas the twist tester rotates the flesh within asingle tissue layer allowing for differences in tex-tural character-istics within that layer to be mea-sured (Griessel and Cr-ouch, 1996).

Soluble solid concentr-ations (SSC) were alsomeasured on pulp tissue using a conventionalrefractonreter (0-30% sugar w/w, Bellinghamand Stanley Ltd., UK). A sample from the mid-dle of the banana was squeezed thlough muslincloth onto the refiactometer glass, aud the SSC(%) recorded after calibration to room tempera-ture.

Arona was neasuled using a l.ìlass spectron-ìe-try-based chemical nose, to determine whetherthere were any characteristic al'oma differences.Pulp (20-30 g) from each of the internal assess-

ment bananas fi-om a single treatment was com-bined and pureed for 6 s in a blender. For eachtl'eatment l0 2O-ml headspace vials were thenfilled with 5 g of the puree and 5 ¡-rl of heptade-cane (an internal standard) was added to thevial. The vials were immediately sealed and ran-dorlly allocated to the Hewlett Packard Chemi-cal Sensor (HP440, Wilmiugton, DE), whichcorlsists of a modif,ed HP 7694 headspace atttosanrpler and a mass sensor based on a HP 5913

MSD. Paraneters for the chemical sensor vi/ere

set as by Muchui (2000).

l8

2.5. Statistical analysis

Tlre first experiment was designed as a 4 x 2

factorial ranclornised complete block with threereplicates. Treatments included the effect ol 0

(air-stolage), 6, 12 or 24 h of nitrogen storagewhen applied to bananas at ripening stage 3

(more green than yellow) or 4 (more yellow thangleen). Harvests on thlee consecutive weeks werereplicales aLrd bananas were ranclomly allocatedto all treatments.

The second experimenl was established in arandomised complete block design with' threereplicates ancl a single treatment. factor., Eachreplicate consisted oF the following nitrogen-stor-age treatnents, 0 (air-storage), 24 h witti a highhurnidity or 24 h with a low humidity during shelflile assessment. All experimental units were t:an-domly distributed to treatments. .. ' ., ,:

Analysis of nosl dzrta (shelf [ile, SSC,;tlmnessand discolouration) was condncted r"rsing the Gen-etal ANOVA directive in Genstat.5 (Release 4.1,4th ecln., 1998, Lawes Agricnltural ,'Trust,Rotharnsted Experimental Station, UK). Strelf lifedata for the first experiment lvere translormed forstatistical analysis by squaring, but were con-vertecl to lhe original units for presentation inTable l. Tleatment eflects were examined forsignificance using a leasl significant difference test(LSD) at the 5olo level.

A. IQieL¡er et ul. / Postlnruesr Biology trtttl Tecltnolog¡' 25 (2002) l5-24

Finally, data on arona were analysed usingstandard principal component analysis (PCA)(Wirthensohn et al., 1999) using the Pirouetteanalysis package (Inlometrix Inc., Woodenville,WA, USA). Each model was validated using across-validation method, which indicated the prin-cipal components that accounted for more thanB0% ol the variation. Fruit with similar volatilecompor-rnds were grouped and separated on plotsfi'om fruit with diffe|ent volatile patterns.

3. Results

3:1. Nitrogen appliccttionfor ucu'ious periods oftime at cliJJerent ripening stcryes

. : . . .1 .

Shelf lile and SSC readings were similar inbananas stored in nitrogen atmospheres at ripen-ing stages 3,or 4 for 6, 12 or 24 h and in bananasstored in air, (Table l). The amount of, surfacediscolouration observed on banana peels variedsignificantly among nitrogen atmosphere treat-ments applied, but incleased in severity withlonger exposure. No slLrlace discolouration was

observed on air-stored bananas; however, brownstreaking developed along tl-re vascltlar tissues iuthe peel of air-stored fruit. This discoloulatiou is

a symptom of mild in-fleld or postharvest chiilinginjury. The injury from nitlogen treatments was

Table I

Assessmenl ol Cavendish bananas cv. Williams at ripening stage 6, alter being stored in a nitrogen attnosplrere lor 0, 6, 12 or 24

lr at ripening stzrge 3 or 4 at 22 "C

Banana ¿rssessn-ìelìt Ripening stage and period of tin-re (h) for which b¿rnanas were stored in uittogen atmosphere

Ripening stage 3 Ripening stage 4

0 6 T2 t2 2424 0 6

Shetf lile (days)"Firmness (kPa)bssc (%)Weight loss (%)

Discolour. index

'7.4¿t 7.4a

l0l a

23.0a2.0a1>

L4b

6.7 ¿

23.2a2.16c2.0c

23.3a2.2c2.7d

23.2it2.tab0.0a

23 5¿

2.labt.4b

7.2aI 04b

23.3a2.0abl.9c

73a 7.3a 7.6a 7.6a

23.|a2.tbc2.8d

23.62t

2.0a0.0a

Datâ are averages of three replicales ¿rnd dillerent letters within a row denote signìfrcant diflerences at P<0.05 using a LSD test.

" Average shelf life (days) data have been back-translormed fror¡ squared analysis data.b Only ripening stages are significant at the 5'% level, therefore the average finlness of bananas stored at r:ipening staþes 3 or 4

were the only nverages given.

A I(licber cr ul f Pttstltctrt'e'tt Biolog' rtnd Tccltnologl' 25 (2002) l5-24 19

Faclo r1

Fig. l. Ttre score plot of barar¡as stored i¡ nitr.ogen for 0 h (air') at stage 3 or' 4 (4, B) at stage 3 for 6' 12' 24 lt (C' D' E) or at

stâge 4 for 6, 12 or 24 h (F' G' H)'

No

oI

clifferent. resltltillg in lar-ge patches of brown dis-

coloLuatioll similal to that fouucl on fruit affected

by iow O, injuly (Wills et ai., 1990; Thompsou'

at stage 3, with lorlgel periods of nitrogen storage

tesultiug in higher weight loss. However, this was

not obselved in fruit relroved at colotlr stage 4

(Table 1).

Fluit analysed at riper-ring stage 6 after stolage

in air or nitrogen fol'variotts pel'iods had differeut

arona characteristics. The PCA models bnilt tocol]-ìpare mass spectlonetric chemical nose data

of all nitlogen-stored as welì as air-stored ba-

llallas explained 91% of the total val'iance present

with two factols, of wl-rich 85% was related to

Factol 1 only. Score plots with the two factor

con-rpoÌ1el.ìts

nitrogen-storl). However,larly those s

have a rlixed aroma- Comparing the plot of the

mass to charge ratios of the ions (variables) that

contlibuteci tã tliis discrirlination (Fig' 2) to the

score plot (Fig. 1), it is clear that ion 55 stt-ongly

correlåted to air--stored bananas, while nitro-qen-

stored frr.tit Inainly correlated to ion 6l'

3.2. Nitrogen crtrtto'sphere storoge in the presettce

o.f lotv or hi.gh relcttioe hLuniditl'

did

n life'

P bo-

tt ttâ

dull yellow peel colour at stage 6' Skin discoloura-

tion ïas sinrilal' iu bananas stolecl in low ol high

lelativeThe 7n/o of the total

valiauc two factors (Fìg'

3). Fac he total v¿rt'iance

with dlstinct discrin-iinatir-rg alorl.l¿ì groupings'

witli one cluster containitlg balrauas stored in air

as well as tlrose stoled ìtr nittoger.r witl-r a low

hlulicitty, ¿rlld the other- cluster consisting of ba-

'¡ ^Æhi

'o "F'þ*.

A

.;].J¡fr"" .'.' " " rqf"'':FÉ

'e'6! cc

È+E€.æ

D

20 A. Klieber et ul. / Posthuruest Biology antl Technolog¡, 25 (2002) t5-24

nanas stored in nitrogen with a high humidity.The clusters containing air-stored bananas andnitrogen-stored bananas in a low hr.rmidity wereclosely associated with iotr 56 (Fig. 4). Ar.omasfrom nitrogen-stored bananas in a high humidityclrrstered around ions 70 and 55. Factor 2 con-sisted of 30% of the total variance. Bananasstoled in nitrogen in a high or low humidity wererelated to ion 6l and 55, whereas air-stored ba-nanas were generally only associated with ion 55(Fig. a).

4. Discussion

Post-climacteric, short-term nitrogen storagedid not extend banana sl-relf life and thereforefurther research into post-ripening tr:eatments toextend sheif life is required. Liu (1976a,b) alsofound that bananas that were initiated with ethyl-ene and then stored in a lo/o O, atmosphere at 14"C ripened soon after being transferred to air at2l "C. This means lhat any reduced O, atmo-sphere eflect is only tlansient once ripening ofbananas has been initiated. In contrast, the gr-een

life of bananas coLrld be extended by short-termlow O, treatments (Wills et al., 1990). [ncreasedethanol and acetaldehyde contents were lound inbananas alter exposure to ethylene, and duringsubsequent continuous low O, exposure, frr-rit ac-cumulated more ethanol and ripening ceased(Imahori et al., 1998). Transient changes were alsolound after short-term low O, exposure of ripen-ing tomatoes (Klieber et a1., 1996), whele it in-duced transient increases in ethanol andacetaldehyde content; howéver', in that case theripening period was extended. Therefore, thereappear to be differences in response to low O,stress based on species, severity of low O, stressand ripeness during application.

However, increasing the sever:ity ol shorl-termlow O' treatments is not an option, as nitlogentreatment in this stucly car.rsed dark discolourationon the peel of fruit. Similar discolouration haspreviously been observed by Wei and Thompson(1993) on bananas stored between 5 and l4o/"COr. Thompson (1998) reported that exposure ofbauanas to < lo/o Oz for an unspecified periodcauses low O, injury in the folm of a dull yellowbr brown peel discolouration, failure to ripen and

NooõL

-0

5

. ,, _. Factorl

Fig. 2. Thd loadings plot of ions (value: mass to charge ratio) stored in nitrogen at stage 3 or 4 for O, 6, L2 or 24 h. Extrsmepositions correlate with similar positioning ol lreatments on the score plot (Fig. l).

61

'86

'Bz

ffir60 = ãg '7't

A. I(liebet et ol. f Po:;rltaruest Biologv und Tecltnolog¡, 25 (2002) l5-24 zl

Table 2

beakel of water oì- without a beakel' of watei' àl 22 "C

Banan¿t ¿ìssessment at ripeningstage 6

Storage of baììanas at lipeningslage 4

Conttol (air') 24 li N, stolage+rvate:: (95%,

RH)24 h N. storage-water (<75%,RH)

Slreli lile (days)

Filmness (kPa)ssc (zù'Weìght loss ('%)

Discolour'. ìndex

70a104.0a

24 2a

17a0.0a

8.0a

103 0a

23.4a2.0a

Llb

74a102.0a

24.0al8a1.4b

Data are avel'ages of three replicates and different letters within a row denote significant dìflerences at P<0 05 using a LSD test

'offl aromas. Wills et al. (1990) also found some

discolouration in nitrogen-treated green fruit.This brown peel discolouration was observed dur-ìng assessment of bananas that had been stored innitlogell for 6 h, and severity increased progres-sively as the stor-age period in nitlogen increased.

A dull peel colour was also observed in the

controls in the second experiment that was con-ducted during winter. The injury is related toslight in-field chilling injury (Marriott, 1980;

Turner, 1997), since fruit wer-e not chilled afterharvest.

Even though there were statistically sigr-rificant

dilfelences in pulp firrnness arnollg banauasstored in nitrogen atn.lospllel'es at ripening stage 3

and 4, these differences would probably not be

detected by consurlers. Tl'ìe largest difference ob-served was less lhan 2o/o of readings, with allbananas being eating soft.

The chemical nose was a simple and reliablemethod for- r'epeated arolr-ìa arìalyses as opposedto lrullan nose detection, which tends to be te-dious for hnman judges (Muclrui, 2000).

The aloma of most of the bananas exposed tonitrogen atrnospheres for the valiorìs peilods oftime was diffelent to the arorna of control ba-nanas stored in air (Fig. 1). The mass to charge

ratios of the ions (variables) that were most in'ì-portant in the discrimination between banana aro-mas had high absolute loading vallÌes, al'rd these

relate to rrìore extreme positions of groups oilscore plots (Fig. 2). Therefore, bananas stored in

air were strorlgiy correlated with an ion with a

mass to cllarge ratio of 55, which arise from the

known banana arolna cotlpounds 3-methylbutylester and l-butanoi that relate to the aroma offully lipe bananas (Malriott, 1980). Pulp frornbananas that were stored in nitrogen were n'iainlycorrelated with an ion with a trass to charge ratioof 61, which alises from ethyl acetate, a banatra

arona componnd that lias au over-ripe arolracharacter. Therefore, the t¡eatments in Fig. 1 thatclustered in between the exttelnes of ion 55 and

ion 61 had a mixed aron-ìa of over-t'ipe and ripe

tones.The presence of ethyì acetate in nitlogen-stoled

banauas at colotu stage 6 may have been due toanaerobic conditions, because ploduction of ethyl

acetate in battanas generally only occtlrs in late

senescence or whell bananas are collsidel'ed inedi-ble (Macku and Jennings, 1987), The initiation ofanaerobic r-espiration in tissues and the tliggeringof feln-ientation reactious, leads to the ploductionof off-odours in fruit (Wills et al., 1998), like the

over-ripe aronla identified in this study. Storage

time in anaelobic conditions would have influ-elìced tlle production of over-ripe aronias, and

this would accourlt for tlie mixed alomas pro-

duced by some nitrogen-stored fruit. Even thoughthe aloma lesults were obtained for bananas at

cololrr stage 6, the nitrogen atmosphere tleatl'ì1el.ìt

nay have tliggered a stl-ess respollse resulting inan increased production of over-ripe al'ollla

cor'ì.ìpourlds,

))

The second experiment had more variablearoma resnlts. The clusters containing air-storedbananas and nitrogen-stored banauas in a lowhun'riclity were closely associated with the ion witha mass to charge ratio of 56 (Figs. 3 and 4), whichis the known banana compotud l-hexanol thatgives an unripe green lone. Aromas ltom nitro-gen-stored bananas in a high humidity clustered

around the ion wilh a mass to charge ol70 that is

largely associatecl with ripe arolna profiles and

compounds like 3-rnethylbr"rtyl ester, [-butanoland propanoic acid; these volatile compounds are

also strongly correlated with ion 55.

Losses in aloma volatiles have been reported inclimacteric fi'uit when stored in low relative hu-mictity (Grierson and Wardowski, 1978), so un-

ripe aromas mentioned previously are iikely to be

duc to water stress preventing the total produc-tion of ripe aromas expected for fully ripe fruit.

Over-ripe volatiles were associated with ba-

nanas stored in nitrogen in a high or low hurnid-ity and this was related lo anaerobic respiration

A. Klieber et trl. f Pttsthurt,est Biology antl Technology 25 (2002) l5-24

inclucing the ploductior-r of alternative aromacompounds. The production of over-ripe volatiles

by lruit at color.tr stage 6, instead of by fruit at

colour stage 7 when lhese volatiles are generally

expected, suggests that an off-odour may have

folmed as a result of nitrogen-stofage and the

stress it caused.

5. Conclusion

Post-ripening storage in a nitrogen attnosphere

priol to commet'cial marketing of bananas did notextend shelf life. This treatment caused skin

browning without delaying tipening or senes-

cence. Although differences in aroma compoundswere found among air-stored and nitrogen-storedbananas when fully ripe, these dillerences did notneceséarily have an adverse effect on flavour' Itwas concluded that storage in a nitrogen atmo-

sphere is not an elfective method f,or extending

the shelf life of ripening bananas.

N

o

6I

Factorl

Fi$. :. fne score plor o[ bananas srored in (A) nitcogen for 0 h (air), (B) 24 h with a low humidity or tor (C) 24 h with a high

humidity.

Ae¡A

B

BA

.B

I.B.B

B

'rb 'ia

'e

'Bqs "E

&B.B

'0""'

c c

'c B

'c

:Ë"

'c'c

'"::'dc

73

Acknowledgements

The autirols wish to thank Chiquita Brands

South Pacific atrd affiliates for their coustaut sup-

polt aud supply of produce. Thanks also to the

Postharvest Departnet.rt of the South Austr-alian

Researci.r and Developmettt Institute for tire tlse

of tl-reil tlvist tester'.

References

Anstralial Banana G¡ower''s Council, 1999 Tlie banaua ltl-dustry in Austlalia, ouline access 8/9/2000 at ltttp://www.abgc olg.au/balana.html.

Courrnonwealth Scientific Industlial Reseal'ch Organìsittion,

1972 Banana Ripening Guide. CSIRO Div Food Res.,

Circular B, North Ryde, NSW, pp. 13.

Glressel. H.M., Crouch, I., 1996 The tolque tester: A newdevelopmerrt to deter'r.ì.rir'ìe liu jt fir'lnness Decid. FrnitGrow 46, 92-99.

Grielsolt. W.. Wardowski, lM.F., 1978. Reìative ìtunicìity e1--

fects on the postharvest lil'e ol- fruìts and vegetables

HortSciencc 1).570-574 ì

H¡rker. F f1., Ir4aindonald, J.H., Jackson, P.J , .199(r. Pcn-

etron-Ieter D1e¿ìsurenrent of apple and kiwifnrit lìnlrncssrìnd instruncnt

23

'86 '57

hlahori. Y, I(ota, M., Ueda. Y., Chachin, K'' 1998 Effects

ol low-oxygen attttosphetes on quaJity ¿rnd etharlol atrd

acetaÌdehyde fortnatiotl ofetllyleue tl'eated ballauas J Jap

Soc Food Sci Tecb¡ol 45,512-576Kliebe¡, 4., Ratalachinakonr, B.' Sinrons, D.H', 1996 Ellect

ol Iow oxygen and higll c¿u'boll dioxide on torl.l¿ìto cultivar

'Berrluda' frtit physiology a:rd conlposition' Sci' l-lort 65'

25r -26t .

Liu, F.W., 1976a. Storing etllylelle-pretreated bauatl¿ls in corl-

tl'olled atnìospliele aud hypobalic air J Atn Soc Hort'

Sci l0l,198-201.Liu, F W , 1976b Banalla respol'ìse 1o lo\j\¡ collcelltl'ations ol

ethyÌerre J, Am. Soc. Hort. Sci l0l' 2??-224'

Macku, C., Jenrlings' W.G.' 1987 Productior.l of voÌatiles by

r-ipenirg banatras. J Agric. Food Clter¡ 35, 845-848'

Malriott. J., 1980. Ban¿rnas - Physiology alrd bioclìerllistry

of storage attd riperling fol optìmunr qrìality CRC Crit'

Rev. Food Sci. Nutr. l3' 4l -88Muchuì, Nt, 2000. The elfect of seasorrs and postharvest

chilÌing on the eating quality oI barlarras Master ol Holti-

ct-tltule, Adelaide UnìversitY,

Rippon, L.E., Trochoulias, T., 1976. Ripening resporrses oi

l:zrnarlas to tenìpel'alure. Aust. J. Exp Aglic' Anir¡ Hllsl¡'

16.140-144Schir:'iL. M, D'hallervi¡, G, Inglese' P, La MLlntia. T" 1999

Epicuticular chattges and storage potential ol cactus ¡reat'

lO ¡ n t t t t i u fi c u s - i n d i t' rt Mt\ler ( L.)l litÌ i t lolì orvin g gib bereì lic

acid prcharvcst spravs trtrd poslhlìrvest lìeât tre¿rtlì.lelll.Postlrtrvest Biol. Technol 17. 79-88.

À. Kliettcr et ul. f Pttsrlttttttesl Biologt' tnd Technologl' 25 (2002) l5-24

0

N

o

GL '98

87"71

Fa cto 11

Fig. 4 The loadi¡gs plot of ions (value : n.ìass [o chalge latio) stoled in nitrogen for 0 h (air')' 24 h with a low htrmidity or for 24

n witl a high humidity. Extrel¡e positìous colrelate with similar positioning of tleatl.ì.ìents otr lhe scole ploL (Fig 3)'

0u4

Operalot'Sci. 121' 927-936.

differences. J. Anr. Soc. Lloitic.

24

Scott, K.J., McGlasson, W.8., Robe¡ts, E.4., t970. Potasstum

permanganate as an ethylene ¿rbsorbent in polyethylene

bags to delay ripeuing of b¿rnanas duting storage. Aust. J.

Exp. Agric. 10, 237 -240.Studmarr, C.J., Yuwana, 1992. Twist test for me¿rsttring lruit

firrnness, J. Text. Stud. 23, 215-227.Thompson. 4.K., t998. Controlled Atmosphere Storage of

Fruits and Vegetables. CAB Intetnational, Oxon.

Tulner, D.W., 1997. Bananas and plantains. In: Mitra, S.K.(Ed.), Postha¡vest Physiology and Storage ofTropical and

Subtropical Fruits. CAB Inreruational, Nedlands, pp.47-83

Wei, Y., Thompson, A.K , 1993. Ivfodifred att-t-rosphere pack-

aging oldiploid bauanas Musa AA. Post-harvest treatment

A. Klieber et al. / Posthru'uest Biology antl Technolttgl' 25 (2002) l5-24

of fruit and vegetables. COST'94 Workshop, 14-t5 Sep-

tenrber 1993, Leuven, Belgiurn, pp.235-246.Wills, R.B.H., Klieber, 4., David, R., Siridltata, M., t990.

Ellect ol brief pre-marketing holding ol bananas in nitro-gen on time to ripen. Aust. J. Exp. Agric.30,579-581.

Wills, R., McGlassou, 8., Graham, D., Joyce, D., t998.

Postharvest: An introduction to the physiology and Lran-

dLing of fuuit, veget¿rbles and ornameutals, 4. Uuiversity olNSW Press, SydneY

Wirthensohn. lvl.G., Francis, I.L., Gawel, R , Jones, G.P.,

1999. Sensory-instrumental correlation of extra vir:gin

olive oil aromas usiug ¿r 'Chemical Nose' Australian Inter-nalional Symposium on Analytical Science, 4-9 July 1999,

Melbourne, Vic., pp. 75-78.

Australian Journal of Experimental Agriculrure, 2002. 42: (in press)

Optimising ripening temperatures of Cavendish bananas var.

''William.s'hanrested throughout the year in QLD, Australia

Bagnato, N., xKlieber, ,A.., Barrett, R. and Sedgley, M.

*Department of Horticulture, Viticulture and Oenology, Adelaide University,'Waite

Campus, Plant Research Centre, PMB. 1, Glen Osmond, S. A. 5064, Australia'

Telephone : (0 S) 8 3 03 66 53, E-mail : andreas.kli eb erfD,adelaide. edu. au

Fax no: (08) 8303 7116

Abstract. Varying bananaripening temperatures were examined throughout the year

to ensure optimum quality and shelf life of commercially ripened fruit in Australia.

Cavendish bananas (var. 'Williams') were harvested throughout the year 2000 and

were ripen ed at 14,16, 18 or 20oC with 300 pL L-l ethylene on 2 consecutive days

until fruit were more yellow than green and then subsequently stored at22"C until the

end of the experiment. Ripening bananas at 14 and 16oC extended shelf life by up to

50Yo and32o/o,respectively. However, ripening bananas at 1'4 or 16"C did increase

peel discolouration, especially on bananas chilled in the field in winter. Bananas

ripened at 18 or 20'C throughout the year had an avenage shelf life of 6 days and

consistently lower peel discolouration. Therefore ripening at 18 or 20"C throughout

the year result in a better visual appearance of the fruit, which is essential for

consumers.

Keywords: temperature, seasons, banana, quality, shelf life

Introduction

Ripe dessert bananas quickly deteriorate in appearance and quality, lasting only a few

days (Israeli and Lahav,1986). Ripening of bananas in commercial practice is

regulated by adjusting ripening temperature, ethylene concentration and humidity to

achieve a suitable ripeness within a planned period of time to satisfy retailer demand.

Suggested banana ripening temperatures differ from l4-21"C (Palmer, 7971), l6-17"C

(Marriott, 1980), l4-24'C (Peacock, 1980), 18-20'C (Salunkhe and Desai, 1984), 16-

21'C (Israeli and Lahav, 1986) and 16-18'C (Turner, 1997). These are all adequate

ripening temperatures for Cavendish bananas (cv. Williams), but may not be optimal'

Optimising ripening temperatures of Cavendish bananas

Palmer (1971) suggests that there is considerable difficulty when predicting the

ripening behaviour of harvested bananas in Australia due to large temperature

variations between seasons. The effect of summer and winter harvesting on optimum

ripening temperatures of bananas in Australia (QLD and NSV/) was briefly

investigated by Young and colleagues between 1929-31(Young et al., 1932), and later

by Rippon and Trochoulias (1976). Young et al. (1932) recommended ripening

temperatures of 20 or 2loC in summer and 19'C for winter. Rippon and Trochoulias

(1g76) agreed that in Australia 19"C was best used for winter bananas, but found that

harvested summer bananas should be ripened at 77oC. However, only two quality

parameters (shelf life and pulp firmness) were measured (Rippon and Trochoulias,

1976). Other important commercial parameters, such as visual peel appearance and

eating quality, should also be measured (Israeli and Lahav, 1986), but were not in the

above study.

This paper reports the effects of ripening tempeiature for harvests in different months

of the year on bananashelf life, peel appearance, pulp firmness, soluble solids and

aroma to allow industry to optimise ripening procedures.

Materials and Methods

Plant Material and Proc e durc

Cavendish bananas var. Williams were harvested from one property in Tully, North

Queensland, and transported at 13"C to the Plant Research Centre (Adelaide

University,'Waite Campus) within three days of harvest. Fruit were harvested atl5o/o

maturity, when green (stage 1) and hard. Evenly sized bananas were dehanded,

deflowered and dipped for one minute in fungicide (0.55 mL L-l Sportak@, Hoechst

Schering AgrEvo, Glen kis, Victoria), then air-dried prior to ripening. After finger

preparation, bananas were randomly allocated to ripening treatments.

Treatments were applied in sealed 10 L plastic containers that contained six banana

fingers, a carbon dioxide scavenger (160 g of Ca(OH)z) and a saturated KNO¡ solution

to maintain a relative humidity of 95%o. Containers were randomly allocated to

monitored, temperature controlled rooms.

2


Effect of ripening temperature on bananra quality

Similar to commercial practice (B. Chartres, Chiquita Brands South Pacific, pers.

comm., 1999),bananas were ripenedat14,16, 18 ot20"C after ethylene gas

injections produced a concentration of 300 pL L-l ethylene in each container. After 24

hrs, the containers were vented for 30 minutes, and ethylene was reapplied.

Thereafter, containers were vented daily, without ethylene application, until fruit were

more yellow than green (stage 4). Bananas were then placed into vented polyethylene

bags at 22"C andassessed daily until fruit started to senesce, showing a yellow peel

colour with light brown flecks (stage 7). This experiment was performed in each of

the months of January, March, May, July, September and November 2000.

Statis tical As s es sment

Every 2 months of the year 2000,bananas were placed at 1 of the 4 temperatures, with

3 different plantation blocks from 1 property acting as replicated, randomised

complete blocks. Every ripening temperature treatment for each replicate contained

12bananafingers; 6 were used for external assessments and the remaining 6 were

used for internal assessments.

Data, except aroma data, were analysed with the Genstat 5 program (Release 4),4th

edition, 1998, Lawes Agricultural Trust, Rothamsted Experimental Station) using the

General ANOVA directive for two factors, harvest date and ripening temperature. A

least significant difference test (LSD) at the 5o/olevelwas used to determine

significant differences between means.

A Pirouette analysis package (Infometrix lnc, Woodenville, WA, USA) using a

Principal Component Analysis (PCA) was used to determine any aroma similarities'

The PCA model formed from banana sample data was cross validated to ensure the

principal components (or factors) accounted for greater than 80% of the variation

between banana samples (V/irthensohn et al.,1999). Therefore samples that were

clustered on plots represented fruit with similar aroma compounds and those that were

separated had dissimilar aromas (V/irthensohn et al., 1999)'

3


Quality assessments

External Assessments

Banana shelf life was recorded by calculating the number of days it took for bananas

to ripen from stage 4 (more yellow than green) to stage 7 (full yellow with light brown

flecks), using the CSIRO banana ripening colour index (CSIRO, 1972). Peel

discolouration was observed visually at ripening stage 6 (full yellow) as follows: 0, no

discolouration present; 1, slight discolouration or dull yellow/grey colouring; 2, mild

discolouration or dull yellow/grey colouring as well as red/brown vascular streaking

along the neck of the banana;3, severe discolouration or grey peel with red/brown

vascular streaking along the length of the peel. Fruit scores (0, l' 2,3) were weighted

and averaged into a discolouration index (DI), using the formula (1) from Schirra er

at. (1999), as data for each treatment was nonnally distributed and fell within a

naffow range. The formula uses the number of fruit with no discolouration (a), slight

(b), mild (c), or severe discolouration (d) compared to the total number of fruit in each

treatment (n).

(a x o) + (å x 1) +(c x 2) +(¿ " z)11¡ ù =

Internal Fruit Assessments

A twist tester (Department of Agricultural Engineering, Massey University, NZ) was

used to measure banana pulp firmness and a hand held refractometer (0-30% sugar

w/w, Bellingham and Stanley Ltd., England) was used to measure soluble solid

concentrations (SSC) of the flesh. All SSC data were adjusted according to laboratory

temperature. Aroma was measured using a chemical nose (HP4440, Hewlett Packard,

V/ilmington, DE) consisting of a modified HP 7694headspace auto sampler on a HP

5973 mass selective detector. A homogenised puree (I20 Ð was taken from the 6

internally assessed bananas for each of the 3 replicates. Ten 5 g samples of the puree

were then measured into 20 mL headspace vials with the addition of an internal

standard, heptadecane (5 pL) prior to sealing. All vials were then randomly placed

into the headspace autosampler for analysis. Headspace sampling parameters were the

same as used by Klieber and Muchui (2002, in press).

4


Results

Daily minimum temperatures were recorded at the South Johnstone research station

for the month prior to harvest (Figure 1, Bureau of Meteorology, 2000-2001). The

months before the July and September harvests show a number of days on which the

daily minimum temperature drops to 13"C or below. Bananas ripened at higher

temperatures (18 and 20"C) in January, May and November had a 16-31% shorter

shelf life than those ripened at lower temperatures (14 and 16"C) (Figure 2). In March

and July, ripening at 14'C resulted in a20-50o/o increase in shelf life, compared to

those ripen ed at 16,18 or 20'C. Bananas ripened at the various temperatures during

September had similar shelf lives of 6 days. Overall banana shelf life ranged from 4-9

days throughout the year and was influenced by ripening temperatures during ethylene

application, as well as harvesting times.

Fruit in January, May and November had a healthy, bright yellow peel appearance

indicated by the low (0 - 0.5) discolouration index scores (Figure 3); these readings

were similar for all ripening temperatures used. Ripening temperatures in March, July

and September did affect banana peel appearance (Figure 3), with ripening

temperatures of 14 or 16'C resulting in more discolouration than 18 or 20"C.

Bananas ripened at20'C were 5-15% firmer than those ripened at l4'C in January,

March and May, but had similar firmness readings in July, September and November

(Figure 4). Firmness readings were similar between most months. Overall, SSC

varied between ripening temperatures, with bananas ripened at 14 or 16"C having a

higher average of 23.0- 23.4% than those ripened at 18 or 20'C'with an average of

22.0%. Monthly trends showed an increase in SSC levels during July and September

(24%) with lower SSC in other months (21.0-22.5%).

Aromas of bananas ripened at 14,16, 18 and20 "C were analysed by mass

spectroscopy at ripening stage 6. The PCA model used to compare these data between

ripening temperatures within and between months explained 92o/o of the total variance

with two factors, where 79o/o and l3o/o related to Factor 1 and 2, respectively. The

majority of the variation between treatments was identified by Factor 1 and so the

results will focus on Factor 1.

5


All bananas ripened in July and September as'well as bananas ripened at 14 and 16'C

in November and those ripened at 14'C in March and May, were located on the

negative side of Factor 1 (Figure 5). All January ripened bananas, as well as those

ripened at 16,18 and 20'C during March and May, and bananas ripened at 18 and

20"C in November, were located on the positive side of Factor 1.

Discussion

Shelf life is an important commercial parameter, but visual appearance is just as

important. Even though ripening temperatures of 14 and 16"C extended shelf life in

most months, the resulting fruit appearance was not always appealing. Low

temperatures slow down respiration and metabolic rates which ultimately extends the

entire ripening and senescence process (Couey, 1982; Wills et al',1998). Rippon and

Trochoulias (1976) reported ripening temperatures 'twere critical in determining shelf

life of bananas harvested throughout the year from NSW, Australia' Holding

temperatures after ripening had little influence on shelf life, although holding

temperatures of 1OoC or higher than the initial ripening temperatures did cause

adverse affects on fruit. Contrary to the previous observations, fruit harvested in

September had similar shelf lives when ripened at 14-20'C. Fruit harvested in

September grew and developed during winter and were affected by field chilling

(Bureau of Meteorology, 2000-2001), which has been found to reduce shelf life of

bananas (Seberry and Harris, 1993). This would account for the similar shelf lives in

September when ripened at l4-20"C compared with other months, when ripening at

14 and 16"C extended banana shelf life by up to 50o/o.

Chilling injury is a physiological disorder of bananas caused by temperatures below

13"C that lowers their visual appearance due to, for example, subepidermal browning

upon fruit ripening (Olorunda et a\.,1978; Israeli and Lahav, 1986; Stover and

Simmonds,1987; Wills et a1.,1998). Subepidermal browning is caused by enzymatic

oxidation of dopamine generally by polyphenoloxidase (Murata, 1969). This accounts

for the high levels of peel discolouration observed in July and September harvested

fruit grown and developed during winter, when field temperatures in the month before

harvest dropped near or below 13'C a total of4 and 9 days, respectively (Bureau of

Meteorology, 2000-200 1 ).

6


Bananas ripened at 14 or 16'C in July and September all had significantly higher

levels of discolouration, compared with those ripened at 18 or 20"C. Lengtþ

development and ripening periods at low temperatures caused an increase in peel

discolouration, compared with that observed on bananas that developed in waÍner

periods of the year, or on those that were ripened at higher temperatures (18 or 20"C).

This is due to a complex time-temperature relationship, where long exposure periods

to temperatures at or near the critical chilling temperature give a similar induced

injury response to that of extremely low temperatures for short periods of time

(Marriott, 1980; V/ills et a\.,1998). Fruit that were stressed prior to ripening and that

were further stressed when ripened atnear chilling temperatures were not able to

recover from the initial chilling exposure period. This explains the high

discolouration index recorded in bananas harvested in July and September when

ripened at above chilling temperatures (14 and 16"C).

March harvested bananas also had higher levels of discolouration when ripened at 14

and 16oC, compared with 18 and2}"C, even though they grew largely during summer

months, and so were not affected by chilling in the held. However, they had been

subjected to wind gusts of 125-170 km hr-l due to a category 2 cyclone only weeks

before harvest (Bureau of Meteorology, 2000), causing visual injury due to excess

physical stress. Peel appearance of March harvested fruit would also have been

influenced by low light levels due to cloud cover during late January and February, a

common occuffence in Queensland. The low light levels may also be influenced by

the use of blue bunch covers in the field, which are generally used to prevent low

temperature stress in banana bunches, but may also prevent light penetration. Low

light levels cause dulling of the bloom (a waxy coating) on bananas, which has a

similar appearance to mild chilling injury symptoms on the peel (T. Johnson, Chiquita

Brands South Pacific, pers. comm., 2001).

Bananas ripened between 14 and 20'C in January, May and November all had

relatively low discolouration indexes, because their development occurred during

autumn, spring or summer when field temperatures were above the chilling

temperature of 13"C.

Ripening temperatures of 14 and 16'C generally resulted in higher SSC levels (I-2%)

and up to 75o/o softer pulp. Similar firmness results were reported by Rippon and

1


Trochoulias (1976), but were not observed by Peacock (1980). Low ripening

temperatures reduce metabolic rates and decreases the rate of respiration (Wills et al.,

1998), consequently extending the time fruit have for textural changes in the pulp as

well as for starch to sugar conversions. Even though SSC levels were significantly

lower when bananas were ripened at higher temperatures, SSC was ahvays above

18%, which is the lowest acceptable level for fully ripe bananas (CSIRO, 1972). Also

consumer detection of these SSC differences would be unlikely, as people differ in

their perception and sensitivity to various foods (Land, 1988).

All bananas analysed had ripe banana aromas, such as fruity esters found in fully ripe

bananas (Marriott, 1980). Horvever, our bananas also had either unripe or over-ripe

aromas at stage 6. Ripe aroma compounds (1-butanol and 3-methylbutyl ester) and to

a lesser extent unripe aroma compounds (2-hexenal) were found to be related to the

negative values of Factor 1, while ripe (butyl esters) and overly ripe aromas (ethyl

acetate) were related to the positive values of Factor I . No off odours or reduced

aroma preferences were found due to mild chilling (Klieber and Muchui,2002), and

therefore varying ripening temperatures of 14-20oC and different harvesting times of

the year are unlikely to have caused aroma acceptability differences. This is in

contrast with severe chilling, where banana aroma is negatively affected (Palmer,

reTr).

Therefore, even though different months of the year and ripening temperatures

resulted in varying banana aroma, this aroma difference does not include unpleasant

bananaaromas and so would not influence consumer acceptability. As banana aroma

acceptability is critical to industry, it was essential for consumer acceptability to be

unaffected by ripening temperatures or harvesting periods, as contrary to ripening

temperatures, harvesting throughout the year cannot be regulated due to continuous

market demand.

V/hile Peacock (1980) found that ripening temperatures between14 and24"C did not

affect banana weight, pulp firmness, shelf life or eating quality, we found that ripening

temperatures of bananas harvested at various times of the year had a substantial effect

on banana shelf life and visual peel appearance. Our results support Rippon and

Trochoulias (1976) and Young et al. (1932) who recommend a ripening temperature

of 19oC for winter harvested fruit. However, our f,rndings do not support the

8


recommendations by Young et al. (1932) of using the higher ripening temperatures of

20 or 2l'C during summer compared to 19'C in winter. Changes in seasonal

temperatures, weather pattems and Cavendish clones since 1932 as well as differences

between the New South Wales and Queensland growing locations may account for the

slight differences in ripening recommendations in 2001. A summer ripening

temperature of 17'C was recommended by Rippon and Trochoulias (1976) which is

similar to our recommendation. However, our results would also support the use of

ripening temperatures as low as 14oC to extend the shelf life of summer harvested

bananas, as long as close monitoring of field conditions occurs throughout the banana

growing period to avoid visual quality reduction by ripening at these low

temperatures.

C-onclusion

Commercial ripening of bananas throughout the year at 18 or 20oC consistently

resulted in fruit with a better peel appearance than ripening at 14 or 16oC. Ripening

of bananas at 14 and 16oC, when fiuit grew and developed over sununer, generally

produced good quality fruit with extended shelf lives, whereas bananas grorwn during

winter and ripened at 14 or 16'C did not. However, the commercial viability of using

14 and 16"C would rely on the constant monitoring of climatic conditions during the

banana growing period, as peel appearance can be negatively affected by sudden

climatic changes such as cyclones.

Acknowledgments

The authors thank Chiquita Brands South Pacific (Adelaide and Queensland) for their

continual supply of fruit and constant support, and the South Australian Research and

Development Institute (Postharvest Group) for their research advice and the use of

their twist tester. Thanks also to Dr Graham Jones and Dr Michelle Wirthensohn for

the use of the chemical nose and for their technical advice.

9


References

Bureau of Meteorology (2000-2001) Climatic data for South Johnstone Research

Station number: 032037, Queensland. Australian Bureau of Meteorology,

Australia.

Couey HM (1982) Chilling injury of crops of tropical and subtropical origin.

HortScience 17, 162-165.

Commonwealth Scientific Industrial Research Organis ation (1972) Banana ripening

guide. cslRo Division of Food Research, circular 8, North Ryde, NSV/, pp.

13.

Israeli Y, Lahav E (1986) Banana. In 'CRC Handbook of Fruit Set and Development'.

(Ed. SP Monselise) pp.45-73. (CRC Press: Boca Raton)

Klieber A, Muchui M. (2002) Preference of banana flavour and aroma are not affected

by time of year or'winter' chilling. Australian Journal of Experimental

Agriculture 42 (in press).

Land DG (1938) Negative influences on acceptability and their control. In 'Food

Acceptability.' (Ed. DMH Thomson) pp.475-483. (Elsevier Science

Publishing: London)

Marriott J (1930) Bananas - Physiology and biochemistry of storage and ripening for

optimum quality. CRC Critical Reviews in Food Science and Nutrition l3r 4l-

88.

Murata, T (1969) Physiological and biochemical studies of chilling injury in bananas.

Physiologia Plantarum 22, 401 -4I1.

Olorunda AO, Meheriuk M, LooneyNE (1973) Some postharvest factors associated

with the occurïence of chilling injury in banana. Journal of the Science of

Food and Agriculture 29r2I3-2I8.

Palmer JK (1971) The banana. In 'The Biochemistry of Fruits and their Products.'

(Ed. AC Hulme) pp. 65-105. (Academic Press: London)

Peacock BC (1980) Banana ripening - effect of temperature on fruit quality.

Queensland Journal of Agricultural and Animal Sciences 37,39-45.

10


Rippon LE, Trochoulias T (1976) Ripening responses of bananas to temperature.

Australian Journal of Experimental Agriculture and Animal Husbandry 16,

140-144.

Salunkhe DK, Desai BB (19S4) 'Postharvest biotechnology of fruits.' (CRC Press:

Boca Raton, Florida)

Schina M, D'hallewin G, Inglese P,LaMantia T (1999) Epicuticular changes and

storage potential of cactus pear lopuntia ficus-indica Mriller (L.)] fruit

following gibberellic acid preharvest sprays and postharvest heat treatment.

Postharvest Biology and Technology 11,79-88'

Seberry JA, Harris DR (1993) 'Factors affecting shelf life of Cavendish bananas.' pp.

387 -39 L (Australasian Postharvest Conference : Gosford, NSW)

Stover RH, Simmonds NW (1987) 'Bananas'. (Longman Scientific and Technical:

Harlow)

Turner DW (1997) Bananas and plantains. In 'Postharvest Physiology and Storage of

Tropical and Subtropical Fruits.' (Ed. SK Mitra) pp. 47-83. (CAB

lntemational : Wallingford)

Wills R, McGlasson B, Graham D, Joyce D (1998) 'Postharvest: An introduction to

the physiology and handling of fruit, vegetables and ornamentals (4th edn.).'

(University of New South'Wales Press: Sydney, NSW)

Wirthensohn MG, Francis IL, Gawel R, Jones GP (1999) Sensory -

Instrumental correlation of extra virgin olive oil aromas using a 'Chemical

Nose'. In 'Proceedings of the Australian International Syrnposium on

Analytical science.' p. 7 5 -7 8. (Australian Intemational Symposium on

Analytical Science: Melbourne, Vic)

Young WJ, Bagster LS, Hicks EW, Huelin FE (1932) 'The ripening and transport of

bananas in Australia.' Council for Scientific Industrial Research, Bulletin No.

64,pp.l-52.

11


^30o9-925J

Ëroo.cblsE210È'-'E5àEo 1 3 5 7 I 11 13 15 17 19 21 23 25 27 29 31

Days of the month

Figure I

*December 1999+April 2000-+August 2000

Chilling point 13'C

-4-February 2000

+June 2000*--E- October 2000

t2


Summer Autumn \ilinter SPring

Cyclone

t\toC"tút,ttGtt0)È

=(,an

2

0

8

6

4

2

0

January March

Figure 2

May July

Month of harvest

Septem ber Novem ber

ø16"Ce20'C

114"Co 18"C

I tsd

l3



Cyclone

3

x(,tc'¿2ofi,

aoolo.9o

0

Figure 3


Month of harvest

September November

114"C Øl 1¡6"C

tr 18"c E2o"c

lsdI

l4

oo-.Y

tt,U'0)

E

_cL

o-

120.00

100.00

80.00

60.00

40.00


I'14"C ø16'C tr 18'C 820'C

January March May July September November

Month of harvest

20.00

0.00

Figure 4

I lsd

15

s.9o(ú

LL


Factorl

Figure 5

Ripe and unripe aroma

"?ßì 'i

'14M

''14M

6JU

'i¿tw¡v .14tvty

'"fru'iîffiqd.,1",.,

Ripe to over-ripe aroma

''t8N1ev

'16M

'18M'18M

t6


Figure captions

Figure 1: The daily minimum air temperatures ("C) from the South Johnstone

Research Station (Bureau of Meteorology, 2000-2001) for the month prior to harvest.

Figure 2: Shelf life of bananas harvested bimonthly and ripened at 14,16, 18 or 20oC

with 300 pL L-l ethylene. Each column represents the mean of 3 replicates with each

replicate consisting of 6 bananas, and the bar denotes least significant differences

between temperatures within months (P<0.05). Climatic events are shown above.

Figure 3: Chilling index of bananas at ripening stage 6 harvested bimonthly and

ripened at 14,16, 18 or 20"C with 300 pL L-1 ethylene. Each column represents the

mean of 3 replicates with each replicate consisting of 6 bananas, and the bar denotes

least significant differences between temperatures within months (P<0.05). Climatic

events are shown above.

Figure 4: Pulp firmness readings of Cavendish bananas at ripening stage 6, harvested

bimonthly and ripened at 14,16, 18 or 2O"C with 300 pL L-t ethylene. Each column

represents the mean of 3 replicates with each replicate consisting of 6 bananas, and the

bar denotes least significant differences between temperatures within months

(P<0.05).

Figure 5: PCA chemical nose score plot of bananas ripened at 14,16, 18 or 20'C in

January (J), March (M), May (MY), July (JU), September (S) or November (N).

t'7

Postharvest Biology and Technolog¡ 2002., (in press).

Research Note

Effect of ethanol vacuum infiltration on the ripening of Cavendish

bananas cv. Villiams

Bagnato, N., Sedgley, M., Bartett, R. and xKlieber, A'

xDepartment of Horticulture, Viticulture and Oenology, The University of Adelaide, Waite

Campus, Plant Research Centre, PMB. 1, Glen Osmond, 5.4.5064, Australia.

Telephone: (+6 1 8) 8303 665 3, E-mail: [email protected]

Fax no: (+618) 8303 7116

Abstract

Bananas have a short shelf life after ripening, with marketers and consumers alike desiring a

longer period before the fruit senesces. To extend shelf life, ripening-initiated bananas were

vacuum-infiltrated with air or ethanol at 10 kPa and subsequently held for 10 min' or 3 h in

the resultant atmosphere. Fruit were then held at 22'C until the conclusion of the

experiment. Shelf life of 4 days and other quality measurements were not affected by the

ethanol treatment as ethanol was only infiltrated into the peel but not the pulp. Therefore,

ethanol vacuum-infiltration is not suitable to extend shelf life of bananas.

Keywords: Musa acuminata Colla, Ethanol atmospheres; Post-ripening; Shelf life; Quality

1. Introduction

Appropriate handling and management of bananas is essential during distribution, as they are

harvested unripe and ripened near their market. An important consideration is that bananas

ripen and then senesce quickly once ripening has been initiated with etþlene (Marriott,

1980; Israeli andLahav,1986). Methods of slowing the onset of ripening of bananas prior to

ripening initiation are controlled atmospheres (Wills et a\.,1982) that induce anaerobic

metabolism and ethanol production (Pesis and Avissar, 1989).

Exogenous application of ethanol has also controlled ripening and senescence of climacteric

fruit (Saltveit, 1989), but not for bananas (Hewage et a\.,1.995; Ritenour et a\.,1997). In the

latter studies, ethanol atmospheres were generated from soaked filter paper in a sealed

container, but neither study reported whether bananas actually absorbed ethanol. A way to

force ethanol absorption into bananas is vacuum infiltration, a technique effectively used by

Ratanachinakom et al. (1999) on tomatoes. The aim of this study was to determine whether

vacuum infiltration of ripening initiated bananas with ethanol atmospheres could extend

bananashelf life without affecting quality and whether ethanol was able to penetrate into

thick skirured fruit such as bananas.

2. Matenals and Methods

Bananas (Musa acuminata Colla Cavendish cv. Williams) of an even maturity (stage 1, hard

and green, CSIRO, 1972) were harvested twice during September 2001, from a single

property in the Tully region of Northem Queensland, Australia. They were transported at 13

oC to the Plant Research Centre (The University of Adelaide, Waite Campus) within three

days of harvest and prepared as described by Klieber et al. (2002).

Ripening of fruit was initiated at 18 'C at 95o/o relative humidity in an atmosphere of 300 pL

L-r ethylene gas in the presence of a carbon dioxide scavenger (160 g of Ca(OH)z). After 48

h, bananas were ripened normally or exposed to a vacuum for 5 min. in a 10 L desiccator (10

kPa pressure) and held after repressurisation in air or over 1 mL of absolute ethanol for 10

min. or 3 h at 22 "C. After treatment periods, bananas were assessed or left to ripen at 18 "C

until they reached ripening stage 4 (CSIRO, 1972). They were then stored at22oC until final

assessments at ripening stage 6 or 7 . Each of the 5 treatments was applied to 2 replicates of

18 fruit. Six of the fruit were used to assess banana shelf life and discolouration, 6 fi:uit were

used to assess frrmness, soluble solid concentrations (SSC), weight loss and ethanol

concentrations in peel and pulp at ripening stage 6, and 6 fruit were assessed for ethanol

concentrations in peel and pulp, immediately after treatments.

Banana shelf life, discolouration, firmness, SSC, and weight loss were assessed as detailed in

Klieber et at. (2002), the hrmness being measured by the use of a twist tester (Department of

Agricultural Engineering, Massey University, NZ). Peel and pulp ethanol concentrations

were analysed immediately after treatment application, as well as after ripening at colour

stage 6, using the method described by Ratanachinakorn et al. (1999). Ethanol

concentrations,ñ/ere analysed using flame ionisation gas chromatography (FID-GC, Varian

3400, Varian Australia, Mulgrave, Victoria), with a stainless steel column (1.8 m x 3.2 mm

OD) packed with Gas Chromt 254 of 80/100 mesh. All data were analysed using general

2

analysis of variance and a least significant difference test (LSD) at the 5o/oleveI, using the

Genstat 5 program (Release 4.I,4rh ed., 1998, Lawes Agricultural Trust, Rothamsted

Experimental Station).

3. Resuls and discussion

Bananas that were or were not vacuum-inhltrated and exposed or not to ethanol atmospheres

had similar pulp firmness of 107- I 13 kPa, SSC of 23.9-245olo, discolouration values of 1 .3-

1.8 and shelf lives of 3.8-4.4 days. Weight loss was slightly higher for control fruit at2.3%o

compared to vacuum-treated fruit at 1.8-2.0% (P<0.05), but this was not visually apparent.

All bananas therefore ripened similarly, with the ethylene-induced ripening process not being

affected. Contrary to our findings with bananas, tomato ripening was slowed by ethanol

vapour application (Ratanachinakorn et al, 1999), by delaying ethylene production (Saltveit,

1e8e).

The reason for this becomes apparent when considering bananapeel and pulp ethanol

concentrations (Table 1). Ethanol concentrations immediately after vacuum infiltration were

seven and 40 times higher at78 and 413 nL g. f. wt.-l, in the peel of bananas held for 10 min

and 3 h in ethanol, respectively, compared to concentrations in the peel of bananas ripened

normally or vacuum-infiltrated with air. However, pulp ethanol concentrations of bananas

measured immediately after vacuum infiltration were similar for all treated fruit. Also

ethanol concentrations of bananas held in ethanol atmospheres were significantly higher in

the peel tissue compared to the pulp tissue of the bananas measured immediately after their

treatment. At colour stage 6, ethanol concentrations of pulp tissues were higher than those

found in peel tissues for all banana treatments, and all were higher compared to levels

immediately after treatment, although no differences were observed between vacuum

treatments.

V/hile vacuum-infiltration with or without prolonged holding forced ethanol into the banana

peel, ethanol did not infiltrate into pulp tissue. This failure to penetrate into pulp tissue was

critical in preventing ethanol-delayed ripening, because pulp tissues play an important role in

ethylene production (Vendrell and McGlasson, 1971). Peel degreening is regulated by pulp

ripening and ethylene production; consequently banana peel colour changes also were not

delayed by applied ethanol (Ritenour et aL.,1997).

3

The high peel and pulp ethanol levels in all ripe bananas can be attributed to the production

of ethanol during the later stages of ripening (Marriott, 1980; Hyodo et a1.,1983; Seymour,

1993;Pesis, 1996). Similar to previous studies, we found that ripening climacteric fruit such

as bananas have initially low ethanol concentrations which increase during the post-

climacteric phase. The normally high ethanol levels during ripening may also explain why

the high peel ethanol levels did not influence ripening. It is, however, also possible that

ethanol declined initially after treatments due to re-metabolisation or volatilisation (Pesis and

Avissar, 1989), thus allowing fruit to start ripening at relatively low ethanol levels, but with

ethanol levels then increasing during ripening.

In summary, vacuum-infiltration with ethanol did not delay ripening of bananas as the

ethanol did not penetrate into pulp tissues, contrary to tomatoes that take up ethanol in the

pulp through the stem scar and the skin. There is scope, however, for testing other indirect

ethanol inducing methods that affect the pulp, such as modified atmospheres'

5. Acknowledgments

The authors wish to thank Chiquita Brands Australia Pty. Ltd. for the supply of all fruit, as

well as the South Australian Research and Development Institute (Postharvest Group) for the

use of their twist tester. Thanks also to the Australian Research Council for funding this

research. Finally a special thanks to Mr Ted Johnson and Dr Edna Pesis for their advice'

References

CSIRO, 1972.Banana ripening guide. Commonwealth Scientific Industrial Research

Organisation Div. Food Res., Circular 8, North Ryde, NSW, pp. 13.

Hewage, K.S., V/ainwright, H., Luo, Y.,1995. Effect of ethanol and acetaldehyde onbanana

ripening. J. Hort. Sci. 70, 51-55.

Hyodo, H., Ikeda, N., Nagatani,4., Tanaka, K., 1983. The increase in alcohol dehydrogenase

activity and ethanol content during ripening of banana fruit. J. Jpn. Soc. Hortic. Sci. 52, 196-

r99.

Israeli, Y., Lahav, E., 1986. Banana. In: Monselise, S. P. (Ed.), CRC Handbook of Fruit Set

and Development. CRC Press, Boca Raton, pp.45-73.

4

Klieber, 4., Bagnato, N., Barrett, R., Sedgley,M.,2002. Effect of post-ripening nitrogen

atmosphere storage on banana shelf life, visual appearance and aroma. Postharvest Biol.

Technol. 254,15-24.

Marriott, J., 1980. Bananas - Physiology and biochemistry of storage and ripening for

optimum quality. CRC Crit. Rev. Food Sci. Nutr. 13, 41-88.

Pesis, 8,., 1996.Induction of fruit aroma and quality by post-harvest application of natural

metabolites or anaerobic conditions. In: Linskens, H. F., Jackson, J. F.' (Eds.), Modern

methods of plant analysis: Fruit Analysis, 18. Springer-Verlag Berlin, Heidelberg, pp. 19-35.

Pesis, E., Avissar, I., 1989. The post-harvest quality of orange fruits as affected bypre-

storage treatments with acetaldehyde vapour or anaerobic conditions. J. Hort. Sci. 64, 107-

1 13.

Ratanachinakorn, B., Klieber, 4., Simons, D. H., 1999. Ethanol vapor vacuum infiltration of

tomatoes: Morphological analysis and effect on ripening and eating quality. J. Am. Soc.

Hortic. Sci. 124, 283-288.

Ritenour, M. 4., Mangrich, M. 8., Beaulieu, J. C., Rab, 4., Saltveit, M. E., 1997.Ethanol

effects on the ripening of climacteric fruit. Postharvest Biol. Technol. 12,35-42'

Saltveit, M. E. J., 1989. Effect of alcohols and their interaction with ethylene on the ripening

of epidermal pericarp discs of tomato fruit. Plant Physiol. 90, 167-114.

Seymour, G.8., 1993. Banana.In: Se¡rmour, G. B., Taylor, J. E., Tucker, G. 4., (Eds.),

Biochemistry of Fruit Ripening, Chapman and Hall, London, pp. 83-106.

Vendrell, M., McGlasson, W. 8.,1971. hhibition of ethylene production in banana fruit

tissue by ethylene treatment. Aust. J. Biol. Sci. 24, 885-895.

V/ills, R. B. H., Pitakserikul, S., Scott, K. J., 1982. Effects of pre-storage in low oxygen or

high carbon dioxide concentrations on delaying the ripening of bananas. Aust. J. Agric. Res.

33, 1029-1036.

5

Table 1: Peel and pulp tissue ethanol concentrations of Cavendish bananas cv. Williams after

ripening with ethylene (300 pL L-t) at 18 oC, treated by vacuum-infiltration with air or

ethanol and held in the resulting atmosphere for 10 min. or 3 h. After the holding period,

bananas were held at 18 "C until they reached stage 4 and ripened at22"C until assessed.

EtOH concentration (nL g.f.wt.-t)'

Immediately after vacuum At ripening stage 6

treatment

Vacuum treatments Peel tissue Pulp tissue Peel tissue Pulp tissue

No vacuum

Vacuum + held for 10min.

Vacuum + held for 3h

Vacuum over EtOH + held

for 10min.

Vacuum over EtOH + held

for 3h

10a

11a

4a

78b

74a

14a

13a

13a

699 a

525 a

484 a

454 a

953 a

800 a

676 a

695 a

413 c 2la 573 a 767 a

' Dataare avefages of 2 replicates where different superscripts in columns denote significant

differences at xP< 0.05 using a LSD test.

6

International Jourrral of Food Science and Technology,2002, (in press).

Quality effects of 1-methylcyclopropene exposure on ethylene-treated

Cavendish bananas

Bagnato, N., Barrett, R., SedgleY,M., and xKlieber, A'

*Postal address: Department of Horticulture, Viticulture and Oenology, Adelaide

University,'Waite Campus, Plant Research Centre, PMB. 1, Glen Osmond, 5.A'5064,

Australia.

Corresponding author's telephone number: (+618) 8303 6653, E-mail address:

andre as. klieb er@,ad e I ai de. edu. au and fax numb er (+618) 8303 7116

Present postal addresses of all authors: same as coresponding author

Abstract

After 48 hrs of ethylene treatment (300 FL L-1) to induce ripening of mature, green

bananas (Musa sp., AAA type, Cavendish subgroup, cv. V/illiams), fruit were exposed

to 0 (non-treated), 3 nL L-l, 300 nL L-r, or 30 ¡rL L-r 1-methylcyclopropene (1-MCP)

for 24,48 or 72hrs at 20'C. Bananas treated with 300 nL L-r 1-MCP had a 6 day shelf

life compared with 3 days for non-treated fruit, and 4 days for fruit treated with 3 nL L-

t l-MCp. lncreased shelf life (half-ripe to over-ripe) occurred without affecting the

green life (unripe to half-ripe) of bananas, peel appearance, pulp texture, soluble solid

concentrations or aroma profiles. Fruit treated with 30 ¡rL L-1 were externally and

internally commercially unacceptable, as fruit developed crown rot prior to ripening.

Application of I-MCP at suitable concentrations could extend banana shelf life, by

enhancing marketing and consumer expectations without compromising banana

quality.

Keywords: 1-MCP, bananas, shelf life, ethylene antagonist, quality

1. lntroduction

Bananas, like other climacteric fruit have a distinct ripening pattern (Marriott, 1980;

Dominguez and Vendrell, 1994; Jíang et a1.,2000). Ethylene, a gaseous plant

hormone, is used in industry to regulate this ripening pattern (Proctor and Caygill,

1985; Seynour, 1993). However, once the ripening process in bananas has been

1

Quality effects of I-MCP on ethylene-initiated bananas

initiated, subsequent deterioration is rapid, which is of concern to marketers and

consumers alike (T. Johnson, Chiquita Brands South Pacific, pers. comm., 2000).

1-MCP is an ethylene antagonist that has recently generated a lot of interest in its use

on bananas. Golding et at. (1998), Jiang et al. (1999a), Jiang et al. (1999b), Harris er

at. (2000), Jiang et al. (2OOO) and Macni sh et al. (2000b) have observed I -MCP effects

on green bananas before ethylene initiation, showing delayed ripening as measured by

colour development, volatile production and fruit softening. Further 1-MCP studies

investigating extension of shelf life (or eating life of ripe fruit) have also been

conducted on bananas that were ethylene-treated prior to 1-MCP application (Macnish

et al,, 1991; Golding et al.,1999; Iiang et al.,1999b; Joyce et al., 1999; Macnish et al.,

2000a). These studies suggest that 1-MCP applied to fruit after ethylene initiation of

ripening extends banana shelf life, but the studies have not supplied suffrcient evidence

to industry and consumers that the quality of fruit at the eating ripe stage is not altered.

Most of the previous reports also did not investigate the application of l-MCP to

bananas after a lengthy period of ethylene initiation (>24hr) that is common in industry

Even though Jiang et at. (1999b) suggest an extension of ripening is only possible

when 1-MCP is applied in the earliest phase of banana ripening initiation, research into

1-MCP application at later ripening stages (>24 hrs after ethylene initiation) has only

briefly been studied by Macnish et al. (1997) and Joyce et al. (1999). Again these

studies do not provide banana quality data necessary for industry and consumers. 1-

MCP use has not been approved on food in Australia or other markets such as the US;

therefore a relatively new sensory instrument known as a chemical nose will be used to

identify potential sensory differences due to banana exposure to I-MCP'

Desirable banana characteristics consist of a ripe product, with a shelf life (days

between ripening stage 4 to 7, Paull, 1996) of >3 days (Australian Banana Grower's

Council, 1999). Therefore, the objective of this study was to examine the external and

internal quality as well as the extension in shelf life due to exposure to various 1-MCP

concentrations and exposure periods applied to bananas 48 hr after ethylene initiation

of ripening.

2

Quality effects of 1-MCP on ethylene-initiated bananas

2. Materials and methods

2.1 Plant møteriøl ønd preparation

Green and hard banana (Cavendish cv. V/illiams) fruit were acquired during January

and February 2001 from a Tullyproperty, North Queensland, and transported at 13"C

to the Plant Research Centre on the Adelaide University Waite Campus. All fruit were

of a closely related maturity, as they were harvested from hands two and three from the

top of the bananabunches. Upon arrival all hands were separated, deflowered, dipped

for 1 min in fungicide (0.55 mL L-r Sportak, Hoechst Schering AgrEvo, Glen kis,

Victoria) and allowed to air dry. Twelve bananas were randomly assigned to each

treatment

Fruit were placed into 10-L plastic containers in the presence of 160 g of Ca(OH)2 (a

carbon dioxide scrubber) and a saturated KNO¡ solution that established a 95o/o relative

humidity. The containers were placed randomly in temperature-controlled rooms at

20"C and banana ripening was initiated with 300 ¡rL L-1 of ethylene gas injected into

the containers on two consecutive days. All containers \¡/ere ventilated for 30 min each

day, for the entirety of the experiment.

2. 2 1 -M ethylcycloprop ene exp o s ure after etlrylen e initicttio n

After 48 hrs of ripening initiation, containers were ventilated and bananas were treated

with 0 (non-treated), 3 nL L-1, 300 nL L-r or 30 ¡rL L-r of 1-MCP at20"c. A 1-MCP

atmosphere was created from Ethylbloc@ (3.3% active ingredient 1-MCP, Rohm and

Haas, Melbourne). Measured amounts of Ethylbloc@ *ere placed into small plastic

cups and placed into the 10-L containers. Reverse osmosis (RO) water at 40oC in a

ratio of 1 g Ethylbloc@ to 16 mL distilled water (G. Baynon, Rohm and Haas, pers.

coÍun., 2000) was added to Ethylbloc@ containers to release 1-MCP. All containers

were sealed immediately upon mixing and placed into 20'C controlled environments.

Fruit were exposed to I-MCP atmospheres for 24,48 or 72 hrs. Containers were

ventilated daily and 1-MCP reapplied as appropriate. At the end of the treatment

period, bananas were removed from containers, placed into unsealed polyethylene bags,

and returned to 20oC for daily assessment until the end of the experiment.

J


2.3 I-MCP quantfficøtion

I-MCP concentration was quantified using aYanan gas chromatograph model 3400

(Varian Australia, Mulgrave, Vic) fitted with a flame ionisation detector and a stainless

steel column (60 cm x3.175 mm id) packed with 80/100 mesh Porapak Q (Alltech,

Sydney, NSW). Column temperature was set at 70'C, while the injector and detector

temperatures \Mere 135'C and 150oC, respectively. Flow rates of air, hydrogen and the

carrier gas nitrogen were 300, 40 and 50 mL min-1, respectively, and the standard gas

used to calculate 1-MCP concentrations was iso-butylene (103 pL L-t) (Jiang et al.,

1999a).

2.4 Bønana assessment

Six fruit from each treatment were used to measure external characteristics, such as

peel colour and discolouration. Banana green life and shelf life were determined

visually by recording the number of days required for bananas to ripen from ripening

stage 1 (green, hard) to stage 4 (peel is more yellow than green) and from stage 4 to

stage 7 (peel is yellow with light brown flecks) (Paull, 1996), respectively. Total

weight loss (%) of fruit was also measured by weighing bananas at ripening stage 1

(peel is hard, green) and 6 (peel is yellow).

The remaining six bananas assigned to each treatment were used to assess internal

quality parameters at colour stage 6. Pulp firmness (kPa) was measured using a twist

tester (Dept. of Agricultural Engineering, Massey University, NZ), because of its

consistency and reliability when measuring soft flesh (Studman and Yuwana,1992).

Soluble solid concentrations (SSC %)'were measured on pulp from the middle of the

banana that had been squeezed through muslin gaùze onto the glass of a conventional

hand-held refractometer (0-30% sugar w/w, Bellingham and StanleyLtd., England).

All refractometer recordings were calibrated to room temperature. Finally banana

aroma was determined using a chemical nose (HP 440, Wilmington, DE) consisting of

a modilred HP 7694 headspace autosampler and a mass sensor based on a HP 5973

mass sensor detector.

Samples of 5 g from a homogenised puree (120 g) taken from the six intemally

assessed bananas and were measured into ten, 20 mL headspace vials. An intemal

standard, heptadecane (5 ¡rL), was also added to each vial prior to sealing. Chemical

nose parameters used were the same as those used by Klieber and Muchui (2002).

4


2.5 Statísticøl analysis

The experiment was established as a 4 x 3 randomised complete block design with

three replicates. Four I-MCP concentrations including 0 (non-treated), 3 nL L-1, 300

nL L-l and 30 pL L-r were applied to bananas for 24,48 or 72hrs. Fruit in weekly

replicates were randomly allocated to treatments. One treatment unit for each replicate

consisted of twelve bananas, with six bananas used for external quality assessments

and the remaining six for internal quality assessments. Analysis of all data was

performed using the General ANOVA command in Genstat 5, (Release 4-1l,4th edition,

1998, Lawes Agricultural Trust, Rothamsted Experimental Station). Data plots were

normalised by transforming total weight loss data into reciprocal data, squaring

firmness data and 1o916 transforming shelf life data. Green life data did not require

transforming. Means, however, have been back transformed for presentation purposes.

A least significant difference test (LSD) at the 5o/olevelwas used to examine all

treatment effects.

Aroma data were analysed using the Pirouette multivariate data analysis package

(lnfometrix Inc, Woodenville, WA, USA), with principal component analysis (PCA).

All data were nonnalised and the PCA model formed was then validated using the

cross-validation method (V/irthensohn et a\.,1999). This indicated the most suitable

factors (principal components) explaining greater than 80% of the variation between

banana samples, with grouped data representing samples with similar volatile profiles

and separating samples for those with dissimilar profiles (Wirthensohn ¿/ al., 1999).

3. Results and Discuss¡on

Exposure periods from24 to 72fus did not affect the efficacy of I-MCP (,Þ0'05, data

not shown). Shelf life, judged as days to ripen from ripening stage 4 to 7 of control

bananas was 3.4 days (Table 1). 1-MCP at3 nLL-l did not increase this shelf life

period, but 30 pL L-t I-MCP completely stopped the ripening of peel and flesh of most

fruit. However, application of 300 nL L-t 1-MCP increased the shelf life of bananas to

6.8 days, while still allowing fruit to ripen.

Green life, pulp firmness and soluble solid readings at colour stage 6 were similar for

bananas treated with 0, 3 nL L-l, or 300 nL L-l I-MCP. V/eight loss readings for

bananas treated with 0, 3 nL L-I, or 300 nL L-1 1-MCP were similarly low, and did not

impact on the visual presentation of the fruit. Therefore, peel and pulp ripening

5


progressed at synchronous rates for these treatments. However, bananas exposed to 30

pL L-l 1-MCP showed significant quality differences compared to all other treatments,

because they had to be examined at ripening stage 2 (peel was green with a trace of

yellow), as fruit did not ripen before fungal spoilage (crown rot) occurred. Firmness

and SSC were 596 kPa and 8olo, respectively, but weight loss (2%) was not dissimilar

to those treated with 0, 3 nL L-r, or 300 nL L-r 1-MCP. Shelf life and aroma could not

be measured as fruits did not ripen.

The process of ripening and senescence of bananas is influenced by their production of

endogenous ethylene. 1-MCP stops ethylene action, including the promotion of further

ethylene production, by binding to the ethylene receptors (Sisler and Serek, 1997). Irr

our experiment, all bananas were ripening-initiated with ethylene before 1-MCP

application. Therefore, endogenous ethylene production and the ripening processes

were in progress when I-MCP was applied.

In bananas treated with 300 nL L-l 1-MCP ethylene action was blocked, slowing down

ripening and senescence without slowing them excessively. The ripening of peel and

flesh of all fruit was synchronous, contrary to Jiang et al. (1999a) who reported uneven

peel degreening in bananas treated with 1-MCP prior to ripening. IJneven degreening

or ripening of fruit is undesirable and would be unacceptable to industry. Ripening

uniformity may have been aided in our experiment by elevating bananas in treatment

chambers to allow all-over gas exchange, as bananas are known to have limited lateral

gas exchange within their tissues (Perez and Beaudry, 1998).

Low concentrations of 1-MCP (3 nL L-1) applied after ethylene initiation were not

successful in extending shelf life. Jiang et al. (1999b), showed that ethylene inhibition

by 1-MCP was non-competitive by measuring the affinity of 1-MCP and ethylene to

binding sites, and observing that 1-MCP had a lower binding constant (K,n) than

ethylene. Therefore, 1-MCP has the greater attraction for ethylene binding sites.

However, there may have been more binding sites than 1-MCP molecules available to

bind to them (Sisler and Serek,1997), or I-MCP did not persist in the tissues to block

the continuously forming ethylene receptors (Jiang et a\.,1999b). This consequently

allowed ethylene to continue to promote ripening.

The etþlene induced ripening process of bananas treated with 30 pL L-l I-MCP was

excessively delayed, with many fruit failing to ripen before crown rot infection reduced

their appearance. The failure of fruit treated with a high 1-MCP concentration (30 ¡tL

6


L-l¡ to ripen, can be explained by limited formation of new ethylene receptor binding

sites (Jiang et al.,I999b), the full occupation of existing binding sites or persistence of

1-MCP in tissues. During the period of ethylene insensitivity and inhibited ripening,

infection of fruit by fungi is not uncommon (Sisler and Serek, 1997), similarly to the

crown rot infections observed on bananas treated with 30 pL L-l 1-MCP. Fruit infected

with crown rot continue to deteriorate as ripening advances (Jones et al., 1993),

reducing banana saleability.

The chemical nose provided reproducible and reliable data, with aroma results

comparable to time-consuming sensory panels (Klieber and Muchui,2002); also as 1-

MCP is not yet approved in Australia, taste panels could not be used. Similar or

dissimilar aroma profiles obtained by the chemical nose give a good indication of

whether consumers are likely to detect differences in aroma between treated and non-

treated bananas.

The PCA model identified 86% of the total aroma variance between treatments to

consist of 2 factors; any remaining factors each made up 3 5%o of the total variance.

Factor 1 explained 75Yo of the variance shown on the x-axis and Factor 2llo/o, shown

on the y-axis (Figure 1). The overall dispersal of treatment data points between Factor

I was found to be similar using a general analysis test. Therefore all bananas analysed

at ripening stage 6 had similar aroma characteristics regardless of 1-MCP level applied,

which can be seen with the overlapping of clustered data values on the plot (Figure 1).

The main aroma compounds identified in the analyses were butanoic acid, butyl ester,

1-butanol, 3-methylbutyl ester and ethyl acetafe, which exhibit banana-like and fruity

aromas (Tressl and Jennings,1972; Marriott, 1980) that are expected from ripe

bananas. Therefore, the application of low concentrations (< 300 nL L-1) of 1-MCP to

ethylene-treated bananas for various exposure periods did not affect the qualitative

composition of banana aromaprofiles obsewed by the chemical nose and so would not

be detected by consumers. Golding et al. (1999) who reported significantly reduced

quantitative production of volatiles using GC-FID in bananas treated'ù/ith 1-MCP,

suggested the reduction in volatile production may affect banana aroma; however, it is

difficult to make any inferences on perceived aroma by consumers using such data, as

consumers tastes and preferences differ.

7


4. Gonclusion

1-Methycyclopropene is a promising postharvest treatment for the extension of banana

shelf life to more than 6 days at 20"C when applied after ripening initiation. Quality of

banana flesh as well as visual appearance was not affected by 1-MCP treatments of

<300 nL L-l. However, the 1-MCP concentration used is critical, as low concentrations

(<3 nL L-1) did not extend bananashelf life and high concentrations (30 pL L-1) caused

fruit to be unacceptable for consumption. I-MCP is not yet approved for use on fruit

and vegetables in Australia, but is approved on food products in Chile and on cut

flowers in the USA. Continual studies are required to assess the effect of various 1-

MCP levels on bananas harvested in different seasons. Larger scale experiments in

commercial enterprises are required to ensure that 1-MCP treatments, when more

widely approved for food use, can be effectively and reliably applied.

Acknowledgments

Special thanks go to Chiquita Brands Australia Pty. Ltd., for supplying all fruit used in

this research and to Mr Ted Johnson for his expert advice. Thanks also to Mr Gregg

Baynon (Rohm and Haas, Australia), Dr Amanda Able and Mr Andrew Macnish for

their technical advice regarding the use of Ethylbloct. A special thanks to Dr Graham

Jones and Dr Michelle Wirthensohn for their guidance on the use of the chemical nose

References

Australian Banana Grower's Council (1999), The Banana Industry in Australia. [V/WV/

do cument] . URL http : //www. ab gc. org. aulb anana. html.

Dominguez, M., & Vendrell, M. (1994). Effect of etþlene treatment on ethylene

production, EFE activity and ACC levels in peel and pulp of banana fruit.

Postharvest Biology and Technology, 4, 167 -177 .

Golding, J. B., Shearer, D., McGlasson, W.8., & Wyllie, S. G. (1999). Relationships

between respiration, ethylene, and aroma production in ripening banana.

Journal of Agricultural and Food Chemistry,47,1646-1651.

Golding, J. B., Shearer, D., Wyllie, S. G., & McGlasson, W. B. (1998). Application of

l-MCP and propylene to identify ethylene-dependent ripening processes in

mature banana fruit. Postharvest Biology and Technology,14,87-98.

8


Harris, D.R., Seberry, J.4., Wills, R. B. H., &. Spohr, L. J. (2000). Effect of fruit

maturity on efficiency of I-MCP to delay the ripening of bananas. Postharvest

B iolo gy and Tecltnolo gy, 20, 3 03 -3 08.

Jiang, Y., Joyce, D. C., & Macnish, A. J. (1999a). Extension of the shelf life of banana

fruit by I-MCP in combination with polyethylene bags. Postharvest Biology

and Technology,16,, I87 -193.

Jiang, Y., Joyce, D. C., & Macnish, A. J. (1999b). Responses of banana fruit to

treatment with I-MCP. Plant Growth Regulation,z&,77-82.

Jiang, Y., Joyce, D. C., & Macnish, A. (2000). Effect of abscisic acid on banana fruit

ripening in relation to the role of ethyl ene. Journal of Plant Growth Regulation,

19,106-111.

Jones, D. R., Pegg, K. G., & Thomas, J. E. (1993). Banana. In: Diseases offruit crops

(edited by D. Persely). Pp.25-35. Brisbane: Queensland Department of Primary

Industries.

Joyce, D. C., Macnish, A. J., Hofman, P. J., Simons, D.H., & Reid, M. S. (1999). Use

of l-methylcyclopropene to modulate banana ripening. In: Biology and

Biotechnology of the Plant Hormone Ethylene 11(edited by A. K. Kanellis, C.

Chang, H. Klee, A. B. Bleecker, J. C. Pech, & D. Grierson). Pp. 189-190.

Dordrecht: Kluwer Academic Publishers.

Klieber, A., 8. Muchui, M. (2002). Preference of banana flavour and aroma are not

affected by time of year or 'winter chilling'. Australian Journal of

Exp erimental Agriculture, 42, 201 -205 .

Macnish, A. J., Joyce, D. C., & Hofman, P. J. (1997).l-Metþlcyclopropene delays

ripening of 'Cavendish' banana fruit. In: Proceedings of the Australasian

P o stharvest Conference, 2 8'h Sept. - 3"t Oct. Pp.282-2 84. Hawkesbury:

University of Westem Sydney.

Macnish, A. J., Joyce, D.C., Hofman, P. J., & Simons, D. H. (2000a). 1-MCP gas can

be used to control banana ripening. Good Fruit and Vegetables, 10r 56'

Macnish, A. J., Joyce, D. C., Hofman, P. J., Simons, D. H., & Reid, M. S. (2000b). 1-

MCP treatment efficacy in preventing ethylene perception in banana fruit and

grevillea and waxflower flowers. Australian Journal of Experimental

Agriculture, 40, 47 l-481.

9


Marriott, J. (1980). Bananas- Physiology and biochemistry of storage and ripening for

optimum quality. CRC Critical Reviews in Food Science and Nutrition,13,4I-

88.

Paull, R. E. (1996). Ethylene, storage and ripening temperatures affect Dwarf Brazilian

banana finger drop. Postharvest Biology and Technology,Sr65-74.

Perez,R., & Beaudry, R. M. (1998). Fractional surface coating modifies gas diffusion

and ripening in bananas. Journal of the American Society for Horticultural

Science,123, 115-1 18.

Proctor, F. J., & Caygill, J. C. (1985). Ethylene in commercial post-harvest handling of

tropical fruit. In: Ethylene and Plant Development (editedby J. A. Roberts, &

G. A. Tucker). Pp. 317-332. London: Butterworths.

Seyrnour, G. B. (1993). Banana. In: Biochemistry of Fruit Ripening (edited by G. B.

Seymour, J. E. Taylor, & G. A. Tucker). Pp. 83-106. London: Chapman and

Hall.

Sisler, E. C. and Serek, M. (1997). Inhibitors of ethylene responses in plants at the

receptor level: Recent developments. Phys iologia Plantarum, 100, 57 7 -582.

Studman, C. J., & Yuwana (1992). Twist test for measuring fruit firmness. Journal of

Texture Studies, 23, 215-227 .

Tressl, R., & Jennings, V/. G. (1972). Production of volatile compounds in the ripening

banana. Journal of Agricultural and Food Chemistry,20,189-192'

V/irthensohn, M. G., Francis, I. L., Gawel, R., & Jones, G. P. (1999). Sensory -

Instrumental correlation of extra virgin olive oil aromas using a 'Chemical

Nose'. Australian International Symposium on Analytical Science, 4-9 July

1999, Melbourne, Vic. Pp. 75-78.

10

Quality effects of I-MCP on etþlene-initiated bananas

Figure 1: Chemical nose data score plot of banana volatiles of fruit at ripening stage 6

after being held for 24 to T2hours in 3 nL L-l l-MCp (4, -),

300 nL L-l I-MCP (8, -

- - ) or in air (C, ""') after ripening initiation.

A

\

III

.2t.

ac

B

tB,\.\

B1

I

II

Factor'1

-10 10

c{

€o

'1

1

1

11


Table 1: Internal and external assessments of Cavendish bananas cv. [4/illiams at

ripening stage 6 (full yellow) following ethylene exposure (300 pL L-r) for 48

hours fruit were subsequently exposed to 0, 3 or 300 nL L-l 1-MCP for 24,48

or 72 hours" at20"C. Fruit were kept at 20"C prior to assessment.

1-MCP concentration

Banana assessment 0 nL L-' 3 nL L-r 300 nL L-'

Green life (days)

Shelf life (days) Y

Firmness (kPa)*

Soluble solid concentration (%)

Total weight loss (%) "

4.1+0.1 a*

3.4+0.1 a

116 a

23a

1.7 a

39+0.2 a

4.3+0.2 a

133 a

22a

1.7 a

3.9+0.2 a

6.8+0.3 b

148 a

22a

2.0 a

' Duration of 1-MCP exposure for all assessments (except shelf life) was not

significant (P>0.05), therefore duration data has been averaged.

v Average data has been back transformed from data that has been log1e transformed.

* Data are means of 3 replicates, where different superscripts within a row denote

significant differences at P<0.05 using a LSD test; * SEM is shown for shelf life and

green life.* Average data has been back transformed from datathat has been squared.

u Average data has been back transformed from reciprocal analysis data.

12

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