prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/6996/1/muhammad_shafique... · iii to the...

Postharvest Pericarp Browning and Quality Management of

Litchi (Litchi chinensis Sonn.)

By

MUHAMMAD SHAFIQUE M.Sc. (Hons.) Horticulture

2003-ag-2438

A thesis submitted in the fulfillment of

the requirements for the degree of

Doctor of Philosophy

In

Horticulture

Institute of Horticultural Sciences

Faculty of Agriculture

University of Agriculture, Faisalabad-Pakistan

2015

II

IN THE NAME OF ALLAH THE MOST BENEFICIENT

AND MERCIFUL

III

To

The Controller of Examinations

University of Agriculture

Faisalabad

We, the supervisory committee, certify that the contents and form of this thesis submitted by

Mr. Muhammad Shafique Regd. No. 2003-ag-2438, have been found satisfactory, and

recommend it to be processed for evaluation by the external examiner(s) for the award of the

degree.

SUPERVISORY COMMITTEE

1) SUPERVISOR: _________________________________

(Dr. Ahmad Sattar Khan)

2) MEMBER: __________________________________

(Prof. Dr. Aman Ullah Malik)

3) MEMBER:

__________________________________

(Dr. Muhammad Shahid)

IV

Declaration

I hereby declare that the contents of the thesis, "Postharvest pericarp browning and

quality management of litchi" are product of my own research and no part has been copied

from any published source (except the references, standard mathematical or genetic models/

equations/ formulate/ protocols etc.). I further declare that this work has not been submitted

for award of any other diploma/ degree. The University may take action if the information

provided is found inaccurate at any stage. (In case of any default the scholar will be

proceeded against as per HEC plagiarism policy).

Muhammad Shafique

2003-ag-2438

Acknowledgement

VI

ACKNOWLEDGEMENT

I indebted to Almighty Allah, the propitious, the benevolent and sovereign whose blessing and glory flourished my thoughts and thrived my ambitions, giving me talented teachers, affectionate parents, sweet brothers and family members and unique friends. Trembling lips and wet eyes praise for Holy Prophet Muhammad (P.B.U.H.) for enlightening our conscience with the essence of faith in Almighty Allah, converging all His kindness and mercy upon him.

I deem it my utmost pleasure to avail myself this opportunity in recording my deep feelings of regards, sense of gratitude, enlightened guidance, affectionate help, personal interest and analytical supervision of Dr. Ahmad Sattar Khan, Associate Professor, Institute of Horticultural Sciences, University of Agriculture, Faisalabad, who always provided necessary facilities throughout this research project. The impression of his kind personality will always remained engraved on my mind.

I offer my appreciations to Dr. Aman Ullah Malik, Professor, Institute of Horticultural Sciences, University of Agriculture, Faisalabad and Dr. Muhammad Shahid, Associate Professor, Deptt. of Biochemistry, UAF, for their ever-inspiring guidance and scholarly suggestions.

I also acknowledge HEC for funding the project No. 2077 entitled “Postharvest storage life and quality management of litchi” under the scheme of “National Research Program for Universities”.

I also acknowledge Institute of Horticultural Sciences and Postharvest Research and Training Center for facilities provided to me for the execution of my research analysis.

I am also obliged to my seniors (Muhammad Amin, M. Shafique Khalid, Omer Hafeez Malik) and my friends (Kashif Razzaq, Sami Ullah, Sajid Ali, Mannan, Adnan, Moin Iqbal, Saeed Akhtar, Adnan Tagga, Ghulam Rasool) who helped me a lot to complete my work successfully.

I am beholden to my parents due to whose prays and wishes, today I am at this position and I do not have words at my command to express my heartiest thanks, gratitude and profound admiration to my esteemed affectionate, loving sisters for their encouragement, immense orisons, mellifluous moral support, patience, spiritual and intellectual inspirations who have always wished to see me glittering high on the skies of success and whose hands always rise in prayer for my success, it is day and night prayer, endurance and ambitious training of my family that brought such a fruit to me. May Allah Almighty infuse with the energy to fulfill their inspirations and expectations and further modify my competence May Allah bless with long happy and peaceful lives (Aameen).

Muhammad Shafique

List of figures

VII

Table of Contents No. Title Page

Acknowledgments……………………………………………………………….. VI Table of contents………………………………………………………………….. VII

List of figures….………….……………………………………………………… X List of tables ………………………………………………....................……… XIV List of symbols and abbreviations…………………………………………….. XV List of chemicals………………………………………………………………. XVII

Abstract ………..….......………………………………………………………. XIX CHAPTER 1 General Introduction…………………………………………................ 1 CHAPTER 2 Review of Literature…………………………………………………….. 5

2.1 Introduction…………………………………………………………………… 5 2.2 Fruit maturity…………………………………………………………………. 5 2.3 Harvest location and cultivars…………………………………………………. 6 2.4 Litchi fruit quality …………………………………………………………….. 7

2.4.1 Soluble solid concentrations (SSC)………………………………… 7 2.4.2 Acidity………………………………….................................... 7 2.4.3 Ascorbic acid …..………………………................................. 8 2.4.4 Total phenolics (TP) …….…………………….......................... 8 2.4.5 Antioxidants………………….…………………................................ 9

2.5 Pericarp browning…………………..………………………………................. 10 2.5.1 Physical causes of pericarp browning……………………………….. 11

2.5.1.1 Water loss………………………………………………….. 11 2.5.1.2 Membrane leakage………………………………………… 12 2.5.1.3 Heat injury ……..……………............................................. 12

2.5.2 Physiological factors affecting pericarp browning….......................... 13 2.5.2.1 Respiration............................................................................ 13 2.5.2.2 Ethylene production……………………………………….. 13

2.5.3 Biochemical causes of browning……..…………............................... 14 2.5.3.1 pH…………………………………………………….......... 14 2.5.3.2 Polyphenol oxidase (PPO)……………………………...…. 15 2.5.3.3 Peroxidase (POD)..………………………………………… 16 2.5.3.4 Superoxide dismutase (SOD)............................................... 17 2.5.3.5 Pigments……………………………………....................... 17

2.6 Management of pericarp browning ………………………………………… 18 2.6.1 Pre-cooling………………….........................……………………… 18 2.6.2 Heat treatment……………………………………………………… 19 2.6.3 Irradiation…………………………………………………………… 22 2.6.4 Storage environment…………………………….…………………. 22

2.6.4.1 Cold storage……………………........................................ 22 2.6.4.2 Controlled atmosphere storage……....................................... 24 2.6.4.3 Modified atmosphere packaging…………………..…........ 24

2.7 Role of different chemicals in pericarp browning……...…….......................... 25

List of figures

VIII

2.7.1 Oxalic acid……………………...………………................................ 25 2.7.2 Ascorbic acid…………………..…………………............................. 27 2.7.3 Hexanal………………………….………………………..….............. 27 2.7.4 Nitric oxide…………………….......................................................... 28 2.7.5 Salicylic acid…………………………………………............. …….. 28 2.7.6 1-MCP……………….……................................................................ 29

2.8 Browning in rambutan…......................................................…………………. 29 2.9 Browning in longan…….……………………………...................................... 30 2.10 Conclusion....................................................................................................... 33

CHAPTER 3 General Materials and Methods………………………………………… 34 3.1 Experimental material…………………………………………………….. 34 3.1.1 Litchi fruit source………………………………………………….. 34 3.1.2 Climate of harvest locations………………………………………. 35 3.2 Storage conditions………………………………………………………… 36 3.3 Parameters studied…………………………………………………………… 36 3.3.1 Physical fruit quality characteristics………………………………. 36 3.3.1.1 Pericarp browning index………………………………… 36 3.3.1.2 Fruit weight loss………………………………………… 37 3.3.2 Chemical characteristics………………………………………….. 37 3.3.2.1 Soluble solid concentrations ……………………………… 37 3.3.2.2 Titratable acidity………………………………………… 38 3.3.2.3 SSC: TA ratio…………………………………………… 38 3.3.2.4 Ascorbic acid……………………………………………. 38 3.4 Determination of total phenolics and total antioxidants…..……………. 39 3.4.1 Total phenolics………………………………………………… 40 3.4.2 Total antioxidants………………………………………………….. 41 3.5 Determination of anthocyanin contents……………………………………… 42 3.6 Determination of activities of antioxidative enzymes……………………….. 43 3.6.1 Catalase…………………………………………………………….. 45 3.6.2 Peroxidase………………………………………………………….. 45 3.6.3 Superoxide dismutase………………………………………………. 46 3.6.4 Polyphenol oxidase…………………………………………………. 47 3.7 Statistical analysis…………………………………………………………….. 48 CHAPTER 4 Harvest Locations and Cultivars Influence Pericarp Browning and

Biochemical Fruit Quality of Litchi fruit…............................................ 49 4.1 Abstract …………………………………............................................ ……. 49

4.2 Introduction…………………………................................................... .…… 49 4.3 Materials and methods …………………………………………………….. 50 4.4 Results ………………………………........................................................ 52 4.5 Discussion …………………………………………………………………. 63 4.6 Conclusions ………………………………..………………………………. 65

CHAPTER 5 Exogenous Application of Oxalic Acid Delays Pericarp Browning and Maintains Fruit Quality of Litchi cv. ‘Gola’………………………. 66

List of figures

IX

5.1 Abstract …………………………………………….................................. 66 5.2 Introduction……………………………………….................................... 66

5.3 Materials and methods …………………………………………………… 68 5.4 Results ………………………………...................................................... 70 5.5 Discussion ………………………………………………………………… 80 5.6 Conclusions ………………………………….………………………….. 83

CHAPTER 6 Postharvest Application of Ascorbic Acid Delays Pericarp Browning and Enhances Antioxidative Capacity of Litchi cv. ‘Gola’ Fruit..….. 84

6.1 Abstract …………………………………………......................................... 84 6.2 Introduction………………………………………………………………... 85

6.3 Materials and methods …………………………………………………….. 86 6.4 Results ……………………………………………………………………. 88 6.5 Discussion …………………………………………………………………. 99 6.6 Conclusions …………………………………………….…………………. 101

CHAPTER 7 Effect of Hexanal on the Storage Life and Pericarp Browning of Litchi Fruit……………………………………………………………… 102

7.1 Abstract ……………………………………................................................ 102 7.2 Introduction…………………………………............................................... 103

7.3 Materials and methods ………………………….…………………………. 104 7.4 Results ………………………………….................................................... 106 7.5 Discussion …………………………………………….…………………… 117 7.6 Conclusions …………………………………………..……………………. 119

CHAPTER 8 Influence of Oxalic Acid, Ascorbic Acid and Hexanal on Pericarp Browning and Antioxidative Enzyme Systems in Litchi cv. ‘Gola’ fruit.................................................................................................. 120

8.1 Abstract …………………………………………………..........................… 120 8.2 Introduction………………………………………....................................… 121

8.3 Materials and methods …………………………………………………….. 122 8.4 Results …………………………….....................................................…… 124 8.5 Discussion …………………………………………………………………. 134 8.6 Conclusions ………………………………..………………………………. 136

CHAPTER 9 Influence of Exogenous Application of Oxalic Acid, Ascorbic Acid and their Combination with Hexanal Fumigation on Pericarp Browning and Quality of Litchi cv. ‘Bedana’ Fruit…....……..………………….. 137

9.1 Abstract ……………………………………………………………….…… 137 9.2 Introduction………………………………………………………………… 138

9.3 Materials and methods …………………………………………………….. 139 9.4 Results ………………………………….................................................... 141 9.5 Discussion …………………………………………………………………. 150 9.6 Conclusions ……………………………………………………………….. 153

General Conclusion and Future Research……………………………………………. 154 References………………………........................................................................………. 158

List of figures

X

List of Figures

No. Title Page Fig. 3.1 Harvest locations: Haripur, KPK (A) and Lahore, Punjab (B)…………. 34 Fig. 3.2 Fruits of two cultivars of litchi used for study ………... 35 Fig. 3.3 Flow chart of extraction procedure for TP and total antioxidants in peel

and pulp tissues of litchi fruit……………..………....................... 39

Fig. 3.4 Flow chart for determination of TP in peel and pulp tissues of litchi fruit…………………………………………………………………………

40

Fig. 3.5 Standard curve of gallic acid for determination of TP in pulp tissues of litchi fruit…………………………………………………………….....

41

Fig. 3.6 Flow chart for determination of total antioxidants in peel and pulp tissues of litchi fruit…………………..…………………………………………….

42

Fig. 3.7 Flow chart of extraction procedure for anthocyanin contents in peel tissues of litchi fruit………………………………………………………………...

43

Fig. 3.8 Flow chart for extraction of antioxidative enzymes in peel and pulp tissues of litchi fruit………………………………………………………………..

44

Fig. 3.9 Flow chart for extraction of polyphenol oxidase enzyme in peel tissues of litchi fruit……………………………………………………………….

44

Fig. 3.10 Flow chart for CAT activity determination in peel and pulp tissues of litchi fruit…………………………………………………………………………

45

Fig. 3.11 Flow chart for POD activity determination in peel and pulp tissues of litchi fruit…………………………………………………………………………

46

Fig. 3.12 Flow chart for SOD activity determination in peel and pulp tissues of litchi fruit……………………………………………………………..

47

Fig. 3.13 Flow chart for PPO activity determination in peel tissues of litchi fruit….. 48

Fig. 4.1 Effect of harvest locations and cultivars on pericarp browning (A, D), anthocyanin contents (B, E) and activity of polyphenol oxidase enzyme (C, F) in peel tissues of litchi fruit during shelf life at ambient conditions……................................................................................... 57

Fig. 4.2 Effect of harvest locations and cultivars on total phenolics (A, C) and total antioxidants (B, D) in peel tissues of litchi fruit during shelf life at ambient conditions………….…………………………………………...

59

Fig. 4.3 Effect of harvest locations and cultivars on ascorbic acid (A, D), total phenolics (B, E) and total antioxidants (C, F) in pulp tissues of litchi fruit during shelf life at ambient conditions…………………………

60

Fig. 4.4 Effect of harvest locations and cultivars on activities of superoxide dismutase (A, D), catalase (B, E) and peroxidase (C, F) enzymes in peel tissues of litchi fruit during shelf life at ambient conditions……………..

61

List of figures

XI

No. Title Page Fig. 4.5 Effect of harvest locations and cultivars on activities of superoxide

dismutase (A, D), catalase (B, E) and peroxidase (C, F) enzymes in peel tissues of litchi fruit during shelf life at ambient conditions……………..

62 Fig. 5.1 Effect of postharvest application of oxalic acid on fruit weight loss (A),

pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase enzyme (D) in peel tissues of litchi fruit during cold storage…………….

71 Fig. 5.2 Effect of postharvest application of oxalic acid on SSC (A), TA (B) and

SSC: TA (C) ratio in pulp tissues of litchi fruit during cold storage…….

73 Fig. 5.3 Effect of postharvest application of oxalic acid on total phenolics (A) and

total antioxidants (B) in litchi peel tissues during cold storage…..

75 Fig. 5.4 Effect of postharvest application of oxalic acid total phenolics (A), total

antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi fruit during cold storage…………………………………………

76 Fig. 5.5 Effect of postharvest application of oxalic acid on activities of superoxide

dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues litchi of litchi fruit during cold storage……………………………………

77 Fig. 5.6 Effect of postharvest application of oxalic acid on activities of superoxide

dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi fruit during cold storage……………………………………………..

78 Fig. 6.1 Effect of postharvest application of ascorbic acid on fruit weight loss (A),

pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase (D) in peel tissues of litchi fruit during cold storage…………………...

90 Fig. 6.2 Effect of postharvest application of ascorbic acid SSC (A), TA (B) and

SSC: TA (C) in pulp tissues of litchi fruit during cold storage…………

92 Fig. 6.3 Effect of postharvest application of ascorbic acid on total phenolics (A)

and total antioxidants (B) in peel tissues of litchi fruit during cold storage………….………………………………………………….

94 Fig. 6.4 Effect of postharvest application of ascorbic acid on total phenolics (A),

total antioxidants (B) and ascorbic acid (C) in pulp tissue of litchi fruit during cold storage…………………………………..

95 Fig. 6.5 Effect of postharvest application of ascorbic acid on the activities of

superoxide dismutase (A), catalase (B) and peroxidase (C) enzymes in peel tissues of litchi fruit during cold storage……………………………

97 Fig. 6.6 Effect of postharvest application of ascorbic acid on the activities of

superoxide dismutase (A), catalase (B) and peroxidase (C) enzymes in pulp tissues of litchi fruit during cold storage……………………………

98 Fig. 7.1 Effect of postharvest hexanal fumigation on fruit weight loss (A),

pericarp browning (B), anthocyanin contents (C) and activities of polyphenol oxidase enzyme (D) in peel tissues of litchi fruit during cold storage……...

108 Fig. 7.2 Effect of postharvest hexanal fumigation SSC (A), TA (B) and SSC: TA

(C) ratio in pulp tissues of litchi fruit during cold storage………………

109

List of figures

XII

No. Title Page Fig. 7.3

Effect of postharvest hexanal fumigation on total phenolics (A) and total antioxidants (B) in peel tissues of litchi fruit during cold storage…………………………………………………………………

111 Fig. 7.4 Effect of postharvest hexanal fumigation on total phenolics (A), total

antioxidants (B) and ascorbic acid (C) in pulp tissue of litchi fruit during cold storage………………………………….. 113

Fig. 7.5 Effect of postharvest hexanal fumigation on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues of litchi fruit during cold storage……………………………………………..

115 Fig. 7.6 Effect of postharvest hexanal fumigation on activities of superoxide

dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi fruit during cold storage……………………………………………..

116 Fig. 8.1 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM

OA + 45 mM AA + 250 µL L-1 hexanal on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase enzyme (D) in peel tissues of litchi cv. ‘Gola’ fruit during cold storage…………


OA + 45 mM AA + 250 µL L-1 hexanal on SSC (A), TA (B) and SSC: TA (C) ratio in pulp tissues of litchi cv. ‘Gola’ fruit during cold storage………………………………………………………………..


OA + 45 mM AA + 250 µL L-1 hexanal on total phenolics (A) and total antioxidants (B) in peel tissues of litchi cv. ‘Gola’ fruit during cold storage………………………………………………………………


OA + 45 mM AA + 250 µL L-1 hexanal on total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi cv. ‘Gola’ fruit during cold storage…………………………………………


OA + 45 mM AA + 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues of litchi cv. ‘Gola’ fruit during cold storage………………………………….


OA + 45 mM AA + 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi cv. ‘Gola’ fruit during cold storage…………………………………


OA + 45 mM AA + 250 µL L-1 hexanal on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase enzyme (D) in peel tissues of litchi cv. ‘Bedana’ fruit during cold storage………

143

List of figures

XIII

No. Title Page Fig. 9.2 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM

OA + 45 mM AA + 250 µL L-1 hexanal on SSC (A), TA (B) and SSC: TA (C) ratio in pulp tissues of litchi cv. ‘Bedana’ fruit during cold storage……………………………………………………………………..

144 Fig. 9.3 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA +

45 mM AA + 250 µL L-1 hexanal on total phenolics (A) and total antioxidants (B) in peel tissues of litchi cv. ‘Bedana’ fruit during cold storage…….…………………………………………………………………….


45 mM AA + 250 µL L-1 hexanal on total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi fruit during cold storage…..


45 mM AA + 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues of litchi cv. ‘Bedana’ fruit during cold storage……………………………………………………


45 mM AA + 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi fruit during cold storage…….....................................................................................................

149

List of symbols and abbreviations

xiv

List of Tables

No. Title Page Table 2.1 Effect of heat treatment on pericarp browning and quality of litchi

fruit………………………………………………………………… 21

Table 2.2 Effect of irradiation treatment on pericarp browning and quality of litchi fruit………………………………………………………….…

23

Table 2.3 Effect of CA /MAP storage conditions on pericarp browning and quality of litchi fruit…………………………………………………

26

Table 2.4 Effect of exogenous application of different chemical on pericarp browning and quality of litchi fruit……………………………………

31

Table 3.1 Climatic characteristics of locations used for the study……………… 36 Table 4.1 Effect of harvest locations and cultivars on weight loss, SSC, TA and

SSC: TA ratio of litchi fruit during shelf period……………………… 54

Table 4.2 Mean effect of harvest locations and cultivars on physiological weight loss, browning index and biochemical quality attributes and enzymatic activities of litchi fruit……………………………………

55

Table 5.1 Relationship of pericarp browning index with activities of oxidative (PPO, POD), antioxidative (SOD, CAT) enzymes, TP and total antioxidants in oxalic acid-treated litchi peel and pulp tissues….………………………………………………………………

72

Table 6.1 Relationship of pericarp browning index with activities of oxidative (PPO, POD), antioxidative (SOD, CAT) enzymes, TP and total antioxidants in ascorbic acid-treated litchi peel and pulp tissues…………………………………………………………………

91

Table 7.1 Relationship of pericarp browning index with activities of oxidative (PPO, POD), antioxidative (SOD, CAT) enzymes, TP and total antioxidants in hexanal-treated litchi peel and pulp tissues……………………………………………

107


xv

List of Symbols and Abbreviations

Abbr. Full name Abbr. Full name ≤ Less than or equal to

°C Degree celsius

% Percent

∆ Changes of

µL L Microlitre(s)

µM Micromolar(s) .

µmol Micromole(s)

1-MCP 1-methylcyclopropene

AA Ascorbic acid

ACC 1-aminocyclopropane-1- carboxylic acid

ACS Acidified calcium sulfate

ADP Adenosine diphosphate

ANOVA Analysis of variance

ATP Adenosine triphosphate

CA Controlled atmosphere

CAT Catalase

CO2 Carbon dioxide / respiration

cv. Cultivar

DPPH 2,2-diphenyl-1-picryl-hydrazyl

EC Enzyme commission

EDTA Ethylenediamine tetra-acetic acid

et al. et alia

FC Folin-ciocalteu

FW Fresh weight

g Gram(s)

h Hour(s)

ha Hectare(s)

HCL Hydrochloric acid

H2O Water

H2O2 Hydrogen peroxide

i.e. That is

kg Kilogram(s)

L Litre(s)

LSD Least significant difference

Ltd. Limited

M Molar

MAP Modified atmospheres packaging

mg Milligram(s)

min Minute(s)

mL Millilitre(s)

mM Millimolar(s)

n Number of sample

Na2CO3 Sodium carbonate

NaHCO3 Sodium bicarbonate

NaOH Sodium hydroxide


XVI

Abbr. Full Name Abbr. Full Name NBT Nitro blue tetrazolium

NO Nitric oxide

NS Not significant

O2 Oxygen

O-2 Superoxide radical

OA Oxalic acid

-OH Hydroxyl radicals

P Probability

pH Symbol denoting hydrogen ion in a solution

POD Peroxidase

PPO Polyphenol oxidase

Pty. Proprietary

PUT Putrescine

® Registered

r Correlation coefficient

RH Relative humidity

ROS Reactive oxygen species

sec Second(s)

S.E. Standard error

SNP Sodium nitroprusside

SOD Superoxide dismutase

SPD Spermidine

SPM Spermine

SSC Soluble solids concentration

TA Titratable acidity

UK United Kingdom

USA United States of America

w/v Weight by volume

List of chemicals

XVII

List of Chemicals

Chemicals Company

For Postharvest application

Ascorbic acid Applichem

Oxalic acid Applichem

Hexanal Sigma

For biochemical analysis

2, 6-dichlorophenol indophenol Fluka

Copper sulphate Applichem

Hydro chloric acid Merck

Lead acetate Riedel-deHaen

Oxalic acid Applichem

Phenolphthalein Merck

Sodium bicarbonate Riedel-deHaen

Sodium hydroxide Merck

Sodium potassium tartrate Riedel-deHaen

Antioxidative enzymes

Di-potassium hydrogen phosphate Applichem

Ethylene diamine tetra-acetic acid Applichem

Guaiacol Across Organic

Hydrogen peroxide Merck

Methionine Merck

Nitro blue tetrazolium Bio-Basic Inc.

Potassium chloride Applichem

Potassium di-hydrogen phosphate Applichem

List of chemicals

XVIII

Chemical Company

Riboflavin Applichem

Triton X Merck

Total phenolic and total antioxidant contents

2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) Sigma

Folin-Ciocalteu (FC) Merck

Sodium carbonate (Na2CO3) Riedel-deHaen

Protein content

2-Mercaptoethanol sigma Merck

Bovine serum albumin (2 x crystallized) MP-Biochemicals

Coomassie brilliant blue G-250 Sigma Merck

Ethanol 95% Merck

Phosphoric acid 85% (w/v) Riedel-deHaen

Sodium dodecyl sulfate Merck

Note:

Riedel-deHaen - Riedel-deHaen, Laborchemikalien GmbH & Co. KG, USA

Chem Alert – Chem alert, Vetnor Avenue, West Perth, Australia

Bio Basic Inc. - Bio Basic Inc. Markham, Ontario, Canada

Applichem – Applichem, Missouri, USA

Fluka - Fluka, Buchs, Switzerland

Across - Across organics, New Jersey, USA

Merck - Merck Pty, Ltd., Lahore, Pakistan

MP Biomedicals - MP Biomedicals India Pty. Ltd, India

Sigma - Sigma-Aldrich Pty. Ltd., Lahore, Pakistan

Abstract

XIX

ABSTRACT

Litchi (Litchi chinensis Sonn.) is an important emerging fruit crop of Pakistan with

good production potential. Rapid pericarp browning is the main postharvest issue which

reduces its cosmetic and market value, limits its extended storage life and causes losses

during its transportation and marketing. This integrated research was planned to check the

influence of cultivars, harvest locations, exogenous application of chemicals such as oxalic

acid, ascorbic acid, hexanal on pericarp browning and fruit quality of litchi under cold

storage conditions. The experiments accordingly followed the experimental designs with

factorial arrangements. The fruit were stored for 28 days with 7 days sampling interval to

determine fruit weight loss (%), pericarp browning index; whereas, soluble solid

concentrations (SSC), titratable acidity (TA), SSC: TA ratio and ascorbic acid were

determined from juice samples obtained from pulp tissues. Moreover, total phenolics (TP),

total antioxidants, activities of peroxidase (POD), catalase (CAT) and superoxide dismutase

(SOD) enzymes were also determined at 7 days interval during cold storage in both peel as

well as pulp tissues of litchi fruit. However, activities of polyphenol oxidase (PPO) enzyme

and anthocyanin contents were determined only from peel tissues. The results of first

experiment suggested that the cultivars and harvest locations significantly influenced

pericarp browning and physico-chemical quality attributes along with the activities of

antioxidative enzymes in litchi fruit. Fruit of litchi cultivar ‘Gola’ exhibited superior quality

characteristics than ‘Serai’; while, litchi fruit harvested from Haripur location showed better

quality than Lahore. Based on the results of the first experiment, litchi cv. ‘Gola’ fruit

produced at Haripur location were selected for rest of the experiments. In second experiment,

postharvest application of oxalic acid was tested on pericarp browning and fruit quality of

litchi cv. ‘Gola’ fruit. Exogenous application of 2 mM oxalic acid delayed pericarp

browning, maintained better physico-chemical attributes along with the higher activities of

antioxidative enzymes (SOD and CAT) in litchi cv. ‘Gola’ fruit during cold storage. Third

experiment was carried out to investigate the effect of postharvest application of ascorbic

acid on pericarp browning and fruit quality of litchi cv. ‘Gola’ fruit. Application of 45 mM

ascorbic acid improved fruit quality by maintaining better physico-chemical attributes and

delayed pericarp browning along with higher activities of antioxidative enzymes (SOD and

Abstract

XX

CAT) in litchi cv. ‘Gola’ fruit during cold storage. In fourth experiment, influence of hexanal

fumigation on the storage life and pericarp browning of litchi cv. ‘Gola’ fruit was

investigated. Hexanal fumigation could not control browning during cold storage period.

Even control fruit showed less pericarp browning than hexanal fumigated fruit. However,

among hexanal treatments, 250 µL treatment showed better fruit quality attributes by

maintaining higher activities of antioxidative (CAT and SOD) and lower activities of PPO

and POD enzymes. From above trials, best concentrations of different chemicals (2 mM

oxalic acid, 45 mM ascorbic acid or 2 mM oxalic acid + 45 mM ascorbic acid + 250 µL

hexanal) were screened and these were re-confirmed on cv. ‘Gola’ along with another

commercial litchi cv. ‘Bedana’. Evidently, 2 mM oxalic acid was more effective in

controlling pericarp browning, improving fruit quality and storage life of ‘Gola’ and

‘Bedana’ litchi fruits, as compared to other chemical treatments. Application of 2 mM oxalic

acid suppressed the activities of pericarp browning enzymes (PPO and POD) and increased

the activities of antioxidative enzymes (CAT and POD). In conclusion, 2 mM oxalic acid or

45 mM ascorbic acid were most effective in delaying pericarp browning and improving

quality of litchi fruit (cvs. ‘Gola’ and ‘Bedana’) under cold storage conditions.

Chapter‐1 General introduction

1

Chapter 1

General Introduction

Litchi (Litchi chinensis Sonn.) belongs to Sapindaceae family and is native to

subtropical areas of southern China and northern Vietnam (Menzel, 2001). Other important

members of this family are Longan and Rambutan. Litchi trees have been cultivated for over

3500 years and the fruit have an important place in Chinese culture. Fresh litchi fruit inspired

many ancient writers and some of the earliest Chinese literature has mentioned about litchi

(Menzel, 2001). In sub-continent, litchi was introduced in the 18th century through Burma,

and from there, it spread all over the sub-continent (Rajwana et al., 2010).

Litchi is a non-climacteric fruit (Wills et al., 2004) famous for its excellent quality

such as juiciness, slightly sour-sweet taste, characteristics pleasant flavour and attractive

pericarp colour. It also has medicinal importance due to physiologically active substances

such as phenol, anthocyanins and flavonoids; which are considered beneficial and effective

in lowering the blood–sugar level and inhibiting hepatitis (Dong et al., 2005). It has high

market value due to deliciously flavoured translucent juicy aril, high nutritive value and

refreshing taste. It contains vitamin C about 27.6 mg/100g FW with substantial amount of

minerals like phosphorus, calcium, iron and is known for presence of antioxidants and

phenolic compounds (Wall, 2006; Hu et al., 2010).

China is the largest producer of litchi in the world; however, it is also produced in

India, Pakistan, South Africa, Israel, Philippines, Brazil, Taiwan, Madagascar, Thailand,

Vietnam, United States and Indonesia (Sivakumar et al., 2007). In Pakistan, litchi is grown

on an area of about 572 ha with annual production of 9250 tons. In Haripur area, the litchi

orchards are spread over 135 ha giving a total production of 180 tons (Shah, 2003). Among

various cultivars of litchi (‘Bedana’, ‘Serai’, ‘Rose’, ‘Gola’, ‘Bombay’, ‘Chinese’, ‘Litchi

Siah’ and ‘Madrasi’) grown in Pakistan, ‘Gola’, ‘Bedana’ and ‘Serai’ are three widely grown

commercial cultivars. Area under litchi cultivation is expanding relatively at slower rate as

compared to its demand in the market, primarily due to high initial costs, long precocity,

shortage of planting material and irrigation water, lack of technical knowledge, improper and


2

poor postharvest handling. Over 90% fruit is domestically consumed as fresh and its export

to foreign countries is nearly negligible which needs to be addressed (Rajwana et al., 2010).

The reddish pericarp of litchi fruit was reported to be 1-3 mm in width (Underhill and

Simons, 1993) with separate translucent aril surrounding a smooth brown seed. The pericarp

surface is covered with numerous protuberances or tubercles (Kumcha, 1998), resulting in a

rough, spiky texture. In some cultivars, such as ‘Wai Chee’, the protuberances flatten at

maturity, resulting in smoother fruit (Batten, 1986). Peel colour varies between cultivars,

from the light orange-pink to deep dull red and the purple-red. The ‘perfect’ litchi may vary

widely in texture, size and shape due to cultivar differences, but, there is one characteristic

which remains constantly desirable i.e. fresh red skin of the fruit. Litchi fruit loses its bright

red peel colour which turns brown within 24 to 48 h after harvest (Zheng and Tian, 2006).

Due to which risk of postharvest losses increase, which are estimated to be around 20 to 30%

and can reach above 50% before reaching to consumers (Wu et al., 2011).

Red peel colour of litchi fruit is one of the major characteristics being used to judge

the commercial quality of litchi. The retention of red fruit colour throughout the postharvest

chain has been remained as a major focus of postharvest research on litchi. Rapid pericarp

browning due to moisture loss reduces market value and causes serious problems in

transportation and marketing of the litchi fruit as pericarp turns brown within few days after

harvest (Huang and Scott, 1985). Although pericarp browning of the skin may not affect the

eating quality of the fruit; it greatly reduces the commercial value in western markets and is

considered one of the major causes of postharvest losses (Snowdon, 1990). This browning

reaction is believed to be induced by desiccation of the pericarp and caused anthocyanin

degradation and the subsequent production of brown by-products (Akamine, 1960). Many

enzymes have been found to regulate pericarp browning in litchi which bring biochemical

and physiological changes, reduction of membrane integrity, degradation of anthocyanins,

increase in peroxidase (POD) and polyphenol oxidase (PPO) activities, and finally enhance

superoxidation reaction (Jiang, 2000). Activities of POD and PPO enzymes degrade red

pigments (anthocyanins) by oxidation of phenolic compounds and form brown coloured

products called o-quinones due to which litchi pericarp turns brown (Zhang et al., 2001a).


3

Besides different other factors, environmental factors including light, temperature,

and moisture contents also have pronounced effects on litchi fruit quality. Previously,

variations in texture of pear and apple fruit were observed among different seasons and

locations (Luton and Holland, 1986; Knee and Smith, 1989). Light is required for proper fruit

development and can improve fruit texture, but light above photosynthetic saturation levels,

especially intense exposure can increase fruit temperature and may result in fruit damage and

a loss of firmness. It is commonly accepted that the temperature during production affects

growth and development. Temperature has a direct influence on metabolism and, thus,

indirectly affects cellular structure and other components which determine texture (Sams,

1999). However, to the best of my knowledge information on potential influence of harvest

locations and cultivars on pericarp browning and quality of litchi fruit is lacking and warrants

investigations.

Commercially SO2 had been used in litchi fruit industry to control pericarp browning,

as it inhibits the formation of brown pigments by inactivating PPO enzyme (Ray et al., 2005).

However, its commercial application has been reduced due to high residual levels and risk of

various health hazardous effects. As an alternative to SO2, oxalic acid has been reported to

delay pericarp browning in litchi at ambient temperature (Zheng and Tian, 2006).

Recently, oxalic acid has gained commercial importance as an anti-browning agent in

litchi and some other fruits (Yoruk et al., 2002). Litchi cultivar ‘Huaizhi’ treated with 2 and 4

mM oxalic acid and stored at room temperature significantly delayed the pericarp browning

due to increase membrane integrity, inhibition of anthocyanin breakdown, decline of

oxidation, and maintenance of relatively low POD activity in the fruit during ambient storage

(Zheng and Tian, 2006). Oxalic acid has been reported to enhance the tolerance in plants by

increasing the activity of POD and producing its isoforms (Zheng et al., 1999). Similarly,

ascorbic acid effectively controlled enzymatic browning of apple (Sapers et al., 1989).

Chitosan combined with ascorbic acid inhibited pericarp browning, dehydration, microbial

attack, maintained membrane integrity and ultimately improved litchi fruit quality (Sun et al.,

2010). Oxalic acid and ascorbic acid when combined with hot water treatment delayed

pericarp browning for 5 days at ambient conditions (Saengnil et al., 2006). Similarly, hexanal

being natural compounds has antimicrobial activity, and it has been reported to extend shelf


4

life of apple slices (Lanciotti et al., 1999). In apple slices it increased aromatic properties by

converting into volatile compounds (Song et al., 1996).

Previously some sporadic work had been conducted to overcome this issue with

inconclusive results. However, an integrated approach had never been adopted to find out

commercially applicable protocol for postharvest management of percicarp browning in

litchi. In addition, the scientific information on commercial cultivars of Pakistan ‘Gola’,

‘Serai’ and ‘Bedana’ regarding postharvest percicarp browning, quality management and

storage life is lacking in the literature. Moreover, the information regarding the difference

between the fruit harvested from different locations is lacking. Therefore, there is great need

to conduct comprehensive studies in order to control percicarp browning and quality

management for these commercially important litchi cultivars of Pakistan. This research will

include series of experiments to improve the internal and external quality of litchi fruit in

order to control pericarp browning and improve fruit quality. Therefore, this study was

planned to develop the novel strategies of improving litchi postharvest storage life and to

achieve the following objectives:

To study the effect of locations and cultivars on pericarp browning and fruit quality

To explore the potential effect of exogenous application of oxalic acid, ascorbic acid

and hexanal on delaying of pericarp browning

To investigate the effects of exogenous application of oxalic acid, ascorbic acid and

hexanal on the fruit quality

To investigate the potential of combine application of above stated chemicals on the

pericarp browning extended storage life and quality of litchi fruit.

Chapter‐2 Review of literature

5

Chapter 2

REVIEW OF LITERATURE

2.1 Introduction

Litchi is a non-climacteric fruit and does not continue to ripe after harvest (Joubert,

1986). It has coarse reddish pericarp with transparent and translucent aril (Underhill and

Simons, 1993). The surface of a pericarp consists of uricles and tubercles resulting in spiky

and coarse texture (Kumcha, 1998). However, some cultivars have smooth pericarp because

of flattening of protuberances at maturity e.g. ‘Wai Chee’ (Batten, 1986). Peel colour of litchi

varies between different cultivars ranging from lite pink to dark red. Litchi fruit varies in

shape, size, and texture but one factor which should remain constant is fresh pink or red

colour of peel as it appeals the consumers. However, litchi fruit due to its perishable nature is

susceptible to physiological damage and deteriorates rapidly within few days after harvest if

kept at ambient temperature. Fruit rapidly loses its bright red skin colour, turning brown

within 1-2 days at ambient temperature (Ray, 1998). The shelf life and visual appearance of

the litchi fruit deteriorates, thereby increasing the incidence of diseases and ultimately affects

the eating quality of the fruit (Mitra and Kar, 2001). This review outlines the historical

progression of litchi postharvest technology with emphasis on effect of low temperature

storage on the physico-chemical characteristics of litchi fruit. Aspects of fruit maturity also

have been included to complete the focus on fruit quality. Following literature briefly shows

the research works previously conducted to study postharvest pericarp browning of litchi.

2.2 Fruit maturity

Litchi fruit being non-climacteric does not ripe after harvest and should be harvested

from the tree when ready to eat. A large number of cultivars are grown so it is very difficult

to set a general guideline for harvesting of litchi fruit (Jiang et al., 2003). Longan fruit loses

its flavour if they are harvested at over mature stage (Wong, 2000), resulting into pulp

hardening (Lin, 2002). In practice, for various cultivars maturity is usually assessed on

characteristics fruit colour and taste. Maturity of litchi fruit can be determined by fruit

weight, colour, SSC, TA, sugar: acid ratio, flavour and day from anthesis. SSC: TA ratio is


6

mostly considered reliable guideline for litchi maturity test. Range of SSC: TA ratio about 40

or greater is recommended for commercial fruit maturity. Batten (1989) found that SSC: TA

ratio is good indicator of flavour, but SSC was not reliable. However, 30-40 SSC: TA ratio is

considered optimum for maturity (Underhill, 1990), whereas, fruit with a SSC: TA ratio of

greater than 80: 1 are considered over mature (Joubert, 1970).

Fruit maturity of various litchi cultivars depends on the fruit colour and taste.

Therefore, harvest maturity and cold storage temperature should be standardized for different

litchi cultivars for their extended cold storage life. Litchi fruit for export should be harvested

at 70% colour development stage and should be held at 5oC during handling, shipping and

marketing. Litchi harvested at 70% colour development stage had an acceptable colour,

flavour and with storage period of 2-3 weeks at 5oC. Similarly, storage life of fruit harvested

at green mature stage was higher than the fruit harvested at fully ripe stage (Shuecheng,

1981). However, maximum orchards are harvested on the basis of taste and overall

appearance. Over-ripe fruit are generally considered sweeter, but exhibit very short

postharvest storage life (Batten, 1989).

2.3 Harvest locations and cultivars

Besides different other factors, environmental factors including light, temperature,

and moisture contents also have pronounced influence on litchi fruit quality. The texture of

the fruit varies with respect to different seasons and locations (Luton and Holland, 1986;

Knee and Smith, 1989). Light intensity has a direct influence on fruit texture as light is

required for proper fruit development and can improve fruit texture. However, if light

intensity exceeds the demand of fruit then it increases internal fruit temperature and may

result in fruit damage. It is commonly accepted that the temperature during production affects

growth and development. Similarly, temperature also influences metabolism, cellular

structure and other components which determine texture of the fruit (Sams, 1999). Texture of

the fruit varies among cultivars within a species with respect to maturity at the time of

harvest (Sams, 1999). European plum cv. 'Green Gage' cultivated at three different locations

in Spain showed significant differences regarding fruit weight loss, colour, firmness and fruit

Ca contents (Guerra and Casquero, 2009). To the best of our knowledge, presently no


7

information is available on the effect of harvest locations on pericarp browning and

quality of litchi fruit with respect to changes in the activities of anti-oxidative enzymes

during ripening and cold storage, which warrants investigations.

2.4 Litchi fruit quality

2.4.1 Soluble solid concentrations (SSC)

SSC play an important role in determining, flavour and nutritive quality of litchi fruit

(Jiang and Fu, 1998). In litchi, SSC of the fruit were increased during ripening, reaching 13–

20% by harvest. Similarly, SSC of litchi fruit was increased significantly during storage from

an initial value of 19.3–23% (Mahajan and Goswami, 2004). Reshi et al. (2014) observed

increase in SSC of litchi cv. ‘Dehradun’ treated with sulphur fumigation and calcium nitrate

dips. Varying behaviour of SSC was also observed by Pornchaloempong et al. (1998) with

increase in storage period. Anoxia treated ‘Huaizhi’ litchi showed increase in SSC initially

but after 6 days of storage decline in SSC was observed (Jiang et al., 2004). On the other

hand increase in SSC was observed when different chemical (sulphur fumigation, borax and

bavistin) were used to treat litchi fruit and then packed in polythene bags (Rajak et al., 2014).

Similarly, Aklimuzzaman (2011) reported increased SSC of litchi cvs. ‘China-3’, ‘Bombai’

and ‘Bedana’ up to 6 days of storage. Mahajan (1997) stored litchi fruit and observed

increasing trend in SSC upto 10 days of storage. SSC of the ‘Calcuttia’ litchi was increased

upto 4 days when treated with different concentrations of alar (50, 100 and 200 mg L-1) and

stored at ambient conditions (Nagar, 1994). Similarly, SSC of ‘Bedana’ and ‘Serai’ showed

continuous decline under cold storage (Khan et al., 2012). Litchi cv. ‘Huaizhi’ treated with

chlorine dioxide showed decrease in SSC during 7 days of storage (Wu et al., 2011).

2.4.2 Acidity

Acidity is an important factor in assessing flavour and nutritive quality of litchi fruit

(Jiang and Fu, 1998). Acidity of the litchi fruit was decreased with extended storage period

(Pornchaloempong et al., 1998). Litchi cv. ‘Huaizhi’ treated with chlorine dioxide showed

remarkable decrease in titratable acidity (TA) during 7 days of storage (Wu et al., 2011).

Concentration of TA decreased during storage due to enhanced respiration and oxidation

activities (Peng and Cheng, 1999). TA of the ‘Huaizhi’ litchi showed decreasing trend when


8

provided with anoxia treatment (Jiang et al., 2004). TA of ‘Bedana’ and ‘Serai’ litchi fruit

showed continuous decline with increase in cold storage period (Khan et al., 2012).

2.4.3 Ascorbic acid

Fresh mature litchi fruit is a significant source of ascorbic acid (40-100 mg/100 g) but

its concentration decrease during storage, regardless of storage conditions (Paull and Chen,

1987). Varying behaviour of ascorbic acid level was observed with extended storage period

(Pornchaloempong et al., 1998). Shah and Nath (2008) found higher initial ascorbic acid in

freshly harvested litchi fruit. The loss in ascorbic acid has been attributed to its high

reactivity to O2 and its degradation has been associated with non-enzymatic browning

(Gimnez et al., 2003). Litchi fruit cv. ‘Huaizhi’ showed decrease in ascorbic acid, when

provided with anoxia treatment (Jiang et al., 2004). Ascorbic acid in ‘Bedana’, ‘China 3’ and

‘Bombai’ litchi decreased with extended storage period (Aklimuzzaman, 2011). Decreased

ascorbic acid in ‘Bedana’ and ‘Serai’ litchi was observed with extended cold storage period

(Khan et al., 2012).

2.4.4 Total phenolics (TP)

Peel of fresh litchi fruit constitutes about 15% of the fresh weight and contains a

significant amount of phenolics (Zhao et al., 2006). There are two types of phenolics found in

different fruit and vegetables i.e. free and bound phenolics. As pericarp browning proceeds,

both types of phenolics decline, especially the free type. These phenolics have antioxidative,

anticancer (Wang et al., 2006) and immunomodulatory properties (Zhao et al., 2007).

However, phenolics are commonly known as the substrates for enzymatic browning of litchi

pericarp. Many discrepancies in phenolic profile of litchi were observed previously. Prasad et

al. (2009) observed epicatechin and epicatechin gallate as major flavonoids present in litchi

pericarp. On the other hand, Zhao et al. (2006) identified procyanidin B2, procyanidin B4

and epicatechin as major flavonoids present in litchi pericarp. Similarly, Liu et al. (2006)

reported epicatechin, pyrocyanidin A2 and A-type pyrocyanidin trimer as major phenolics

present in litchi pericarp. There are differences among scientists about the amount of

phenolics present in litchi pericarp as TP reported by Ruenroengklin et al. (2008) were 6-fold

different then the amount of phenolics reported by Prasad et al. (2009). TP were decreased

gradually in the peel and pulp tissues of litchi fruit (Wu, 1995). Contents of total phenols


9

decrease with increase in storage period (Peng and Cheng, 1999), but under optimum storage

conditions higher level of TP were maintained (Chen and Hong, 1992). TP of ‘Bedana’ and

‘Serai’ were decreased continuously when stored at 5oC for 28 days (Khan et al., 2012).

2.4.5 Antioxidants

All organisms contain antioxidative defense mechanism which protects them from

oxidative damage (Simic, 1988). Antioxidants are molecules that slow down the oxidation by

termination the oxidation chain reaction (Yanishlieva et al., 2006). There are numerous

antioxidants which differ in their rate of activity, such as ascorbic acid, glutathiones,

carotenoids, tocopherols, polyphenols and many other phytochemicals, which protect plants

from oxidative damage by preventing the production of reactive oxygen species (ROS)

(Bloknina et al., 2003; Seifried et al., 2007) and are very efficient in controlling enzymatic

browning in pineapple slices (Gonzalez-Aguilar et al., 2005). Litchi fruit contains significant

amount of antioxidants like other fruit (Isabelle et al., 2010) which helps litchi fruit to

maintain its bright red colour (Duan et al., 2007b). Among antioxidative compounds,

ascorbic acid is the most common antioxidant present in different fruit and vegetables, while

polyphenols are an important group of antioxidants present in plants because of their diverse

and extensive distribution. Polyphenols possess the ability to scavenge both electrophiles and

ROS (Robards et al., 1999). Different antioxidants like isoascorbic acid, N-acetyl cysteine

have been found very effective to reduce the browning and prevent the postharvest decay of

fruits and vegetables. Activity of polyphenol oxidase (PPO) enzyme can be decreased

effectively by the use of isoascorbic acid and N-acetyl cysteine than other antioxidants like

glutathione and citric acid (Lei et al., 2004). On the other hand, salicylic acid (C7H6O3) has

been reported to control number of biochemical processes taking place in plants. Salicylic

acid also has an antagonistic effect on ethylene biosynthesis and/or ethylene action (Raskin,

1992). Salicylic acid (0.5%) effectively inhibited PPO activity, controlled pericarp browning

and improved anthocyanin contents of litchi fruit (Kumar et al., 2013). In another study

substrates (epicatechin and procyanidin A2) for PPO were extracted from litchi peel tissues

to identify the antioxidative potential. It was found that epicatechin contents decrease with

increase in pericarp browning at ambient conditions (Sun et al., 2010).


10

2.5 Pericarp browning

The commercial quality of litchi is described by its peel colour. Therefore,

postharvest research on litchi is mainly focused on retention of its fresh red colour. Litchi

fruit is highly perishable, deteriorates rapidly and loss red peel colour within three days after

harvest which ultimately affects its cosmetic value (Huang and Scott, 1985). Pericarp

browning is believed to be caused by rapid degradation of the red pigments by PPO enzyme,

forming brown-colored byproducts (Huang and Wang, 1989; Jaiswal et al., 1987). Red

pigments in the litchi pericarp deteriorate rapidly by the action of PPO enzyme, thereby,

producing brown coloured by products called o-quinone giving brownish appearance to the

pericarp of the fruit (Jiang et al., 2004). Skin browning initiates from the protuberances of the

pericarp and consequently covers the whole surface of pericarp, ultimately pericarp become

dehydrated and brittle (Underhill and Critchley, 1995). Mesophyll cells of the pericarp turns

brown first followed by epicarp and endocarp (Joubert and Van Lelyveld, 1975). Therefore,

affecting its commercial value in foreign markets and finally affecting eating quality of the

fruit due to peel browning which increases the postharvest losses (Snowdon, 1990).

Enzymatic browning in litchi fruit is a major problem leading to undesirable fruit

characteristics and decrease fruit quality (Jiang and Li, 2003). Postharvest pericarp browning

of litchi is mainly due to degradation of pigments and oxidation of phenolics by PPO and

peroxidase (POD) enzymes (Zhang and Quantick, 1997; Jiang, 2000; Zhang et al., 2005).

Different stresses are involved in pericarp browning of litchi such as climatic conditions at

the time of harvest, chilling injury (Tongdee et al., 1982), desiccation (Underhill and Simons,

1993; Lin, 2002), diseases (Jiang et al., 2003), heat injury (Jiang, 2002), micro-cracking,

senescence and decay (Underhill and Critchley, 1993). Other factors, like injury during

harvest, pathogen attack and storage of fruit at undesirably low temperatures. Water soaked

dark lesions appear on pericarp if browning is due to temperature stress, whereas, desiccation

browning causes pale and dry appearance on pericarp (Bhushan et al., 2015). Pericarp

browning does not affect the eating quality of fruit; however, it deteriorates the cosmetic

value of fruit as costumer avoids buying brown and desiccated fruit (Fitzell and Coates,

1995).


11

2.5.1 Physical causes of pericarp browning

2.5.1.1 Water loss

Dehydration or moisture loss is major cause of postharvest losses in horticultural

industry. Symptoms of moisture loss and its extent vary with kind of fruit and their

characteristics. Moisture loss symptoms may include pericarp browning, dry appearance,

softening, shriveling and loss of crispiness. In general, symptoms of moisture loss appear

when weight loss of the fruit reaches to 4-8% (Van den Berg, 1987). Moisture loss causes

decrease in weight and decline in appearance which ultimately leads to pericarp browning of

litchi (Scott et al., 1982). The damage due to water loss can be reversed if fruit are placed in

high humidity conditions, but irreversible damage takes place if water stress is prolonged

(Shewfelt, 1993). Moisture loss causes desiccation of the peel, thereby, deteriorating the

fresh red colour of litchi fruit and giving it pale brown appearance. Mechanical injury cause

increase in moisture loss and respiration rate due to which sensory quality and shelf life of

the fruit is reduced (Wills et al., 1998). Conductivity of litchi pericarp tissue and membrane

integrity is associated with water loss. At 10% moisture loss, conductivity of litchi pericarp

was ranged from 50-100 μS to 100-50 μS and this conductivity was reached to 150-250 μS at

20% moisture loss. Therefore, it is often said that pericarp browning in litchi is due to

moisture loss (Chen and Hong, 1992). However, there is contradiction among scientists about

the moisture loss required to cause pericarp browning in litchi as Underhill and Critchley

(1994) reported that even as little as 2% moisture loss is enough to cause pericarp browning

in litchi. In another study, weight loss upto 3-5% caused pericarp browning in litchi fruit

(Brown, 1986). On the other hand Liang et al. (1998) reported that browning was initiated at

7.5% weight loss and fruit did not turn brown completely until fruit weight loss reached to

18%. Similarly, Shi et al. (2001) reported that pericarp browning in litchi fruit was started

when weight loss reached to 9%. This difference about the amount of weight loss required to

trigger pericarp browning may be due to different browning assessment methods, cultivars,

condition of orchard, fruit moisture content at the time of harvest and postharvest handling

methods. Browning due to moisture loss initially starts from one side of the pericarp and

progressively covers the entire pericarp, which becomes pale brown, coarse and brittle

(Underhill and Simons, 1993).


12

2.5.1.2 Membrane leakage

Browning of plant tissues is caused by membrane degradation (Toivonen, 1992). The

accumulation of malondialdehyde showed that fruit were senescing. Membrane leakage was

increased gradually during storage as degree of senescence and injury of fruit was increased

(Gao et al., 1990). Membrane integrity was decreased with increase in storage period which

caused leakage from the membrane (Marangoni et al., 1996). Dipping of litchi fruit in

adenosine triphosphate (ATP) solution resulted in less relative leakage with reduced pericarp

browning index (Song et al., 2006). In another study it was observed that calcium contents in

the litchi pericarp were negatively correlated with the rate of membrane deterioration (Huang

et al., 2003). Low temperature increased the rate of membrane leakage; however, low

temperature and storage time showed different effects on different cultivars (Wang et al.,

1996). Oxalic acid significantly reduced the relative leakage rate, as compared to control fruit

when kept at ambient conditions (Zheng and Tian, 2006). Salicylic acid effectively reduced

membrane leakage of litchi fruit (Kumar et al., 2013).

2.5.1.3 Heat injury

Litchi pericarp browning is a general surface injury commonly observed after harvest

(Nip, 1988). Injury is known to occur in response to a wide range of factors, including

climatic conditions during ontogeny fungal infection, desiccation, fruit senescence, and heat

injury. Once present, it is difficult to identify the kind of stress that caused injury (Bryant,

2004). As a result of this seemingly uniform injury, litchi pericarp browning has been studied

as a single degradation system, with little emphasis placed on the original stress. Previously,

most of the researchers targeted pericarp browning caused by postharvest desiccation.

Relatively little is known about injury mechanisms under other conditions, or their similarity

to ambient desiccation browning. Browning is commonly attributed to degradation of the

anthocyanin pigments by PPO enzyme activity (Akamine, 1960). However, it is not clear

whether the by-products of this degradation contribute to tissue browning. Mature litchi fruit

were heat-treated at 60°C for 10 min to study heat-induced pericarp browning. PPO enzyme

activity of the litchi pericarp was increased immediately with rapid anthocyanin degradation

and tissue browning was observed 2 min after heating and pigments were distributed

uniformly throughout the pericarp. The distribution of brown pigments was different than the


13

highly localized browning observed under ambient desiccation. Although both ambient and

heat-induced pericarp browning are visually similar but the anatomical distribution of brown

pigmentation is quite distinct. The distribution of brown pigmentation was not consistent

with anthocyanin localization. Following ambient desiccation, the mesocarp became

colourless even though this represented the greatest concentration of pigment. Browning

caused by heating may result from non-selective degradation of a range of compounds,

including anthocyanin (Peng and Cheng, 1999).

2.5.2 Physiological factors affecting pericarp browning

2.5.2.1 Respiration

Litchi fruit being non-climacteric showed no significant differences in respiration

peak, which is very important factor in determining the storage life of fruit. However,

respiration rate differs with type of storage conditions. For example, litchi fruit kept at

ambient conditions released CO2 up to 200 mg kg-1 h-1 and fruit under cold storage released

CO2 about 60-80 mg kg-1 h-1 (Chen et al., 1987). This change in the respiration rate is very

important factor hampering the long-term storage. The high respiration rate may result in

rapid consumption of nutrients, leading to a loss of taste after storage. The respiration rate of

litchi fruit stored at room temperature was three times higher than that in cold storage (Hong

et al., 1986). The respiration rate declined at the early stage of storage but increased during

long term storage as pericarp turned brown (Gao et al., 1990). However, respiration rate of

litchi fruit was inhibited by low temperature storage at 4oC (Huang et al., 1990). During the

course of storage at low temperature, the respiration of litchi fruit was declined. After the

fruit being transferred out from the cold storage, the temperature of fruit increased, resulting

in a gradual browning of pericarp. This may possibly be one of the reasons for the short

shelf-life of fruit (Huang et al., 1990).

2.5.2.2 Ethylene production

As a non-climacteric fruit, litchi exhibits low ethylene production. Peak ethylene

production was observed in the middle stage of development and declined gradually (Ji et al.,

1992). However, some researchers observed no influence of ethylene on the ripening of the

fruit (Jiang et al., 1986). The exogenous application of silver thiosulphate (STS) inhibited


14

ethylene production in the fruit, retarded the growth, and resulted in negative effects such as

delaying the colour development of the fruit and excessive fruit-drop with cracking (Yi et al.,

1999). In normally matured fruit, the amount of ethylene released was less than over-ripe

fruit (Chen et al., 1986). Ethylene production during 3-5 days of storage at ambient

conditions was considerably high during initial days of storage and later on decreased

gradually. On the other hand, storage of litchi fruit under low temperature conditions was

useful in inhibiting ethylene production (Jiang et al., 1986). When fruit were transferred to

20oC from cold storage, ethylene and ACC both showed an increase at 5°C and 8°C,

whereas, fruit stored at 1°C, showed lower level of ethylene and ACC than fruit stored at 5°C

and 8°C. These results suggest that storage at 1°C resulted in chilling injury of litchi fruit

(Huang et al., 1990). On the contrary, fruit stored at room temperature had a lower level of

ethylene production with no obvious peak (Liang et al., 1998). When fruit were transferred to

room temperature after storing at low temperature, the ethylene production increased rapidly

and reached a peak at 14 h before it decreased. The tendency of ethylene change is similar to

that of the respiration change (Jiang et al., 1986).

2.5.3 Biochemical causes of browning

2.5.3.1 pH

The changes of pH value in the pulp or peel tissues of litchi fruit have significant

effect on red color or anthocyanin contents and the activities of enzymes in the peel/pulp

tissues. The pH values of both peel and pulp tissues increases gradually with increase in

pericarp browning (Wu, 1995). However, storage conditions also influence the pH value.

Litchi fruit stored at ambient temperature for 6 days showed substantial increase in pH from

4.3 to 5.3, whereas, at low temperature storage smaller increase (4.3 to 5.0) in pH was

observed (Hong et al., 1986). Similarly, activity of PPO enzyme was gradually increased

with increase in pH causing pericarp browning of litchi fruit (Underhill and Critchley, 1995).

Among different other factors, moisture loss or dehydration has direct effect on pH

and activity of PPO enzyme as significantly higher activity of PPO enzyme was observed at

7-7.4 pH, whereas, no activity was observed 4.2 pH. Desiccation browning raises pH in the

vacuole and slight increase in pH from acidic to basic is sufficient to degrade anthocyanin

contents. At ambient conditions (25oC and 60% RH), slight increase in pH from 4.15 to 4.52


15

decolourized anthocyanins and were sufficient to cause pericarp browning within 48 h after

harvest (Underhill and Critchley, 1994). Increase in moisture loss causes increase in pH. This

increase in pH enhances the activity of PPO enzyme and cause pericarp browning. In litchi

fruit, 7.4 pH was effective enough to activate PPO enzyme and cause pericarp browning

(Jiang et al., 1997). However, the red colour of pericarp can be restored temporarily by use of

acid dipping which shows the influence of pH on anthocyanin pigments (Underhill and

Critchley, 1994). Therefore, it can be said that anthocyanin are under the direct influence of

pH as ratio between anthocyanin pigments molecules changes with varying pH and the red

colour of anthocyanin decreases with increase in pH (Brouillard, 1982). Therefore, acidic

solutions are required for anthocyanin to re-express the red colour. Similarly, Pang et al.

(2004) also reported that anthocyanin contents were decreased with increase in pH. It can be

said that epicarp browning can be covered by the mask of red anthocyanin pigments through

acid dipping of fruit. Low pH also increased the stability of anthocyanin, and resulted in red

colour of the litchi fruit pericarp (Tongdee, 1998), as activity of PPO enzyme was inhibited

at low pH and ceased at 4.2 pH (Jiang et al., 1997). Therefore, to inhibit PPO activity pH

should be maintained acidic (Underhill and Critchley, 1994).

2.5.3.2 Polyphenol oxidase (PPO)

It is believed that activity of PPO is responsible for the degradation of anthocyanin

and other phenolic compounds in many fruit species (Mathew and Parpia, 1971). Similarly, it

is widely presumed that in litchi fruit anthocyanin breakdown and pericarp browning is due

to the increase in activity of PPO enzyme (Akamine, 1960). Total phenolics concentration

and PPO enzyme activity are correlated in litchi pericarp browning. As activity of PPO

enzyme increases, it oxidizes the TP present in litchi pericarp and cause browning (Jiang and

Fu, 1998). Increased activity of PPO enzyme causes different processes including

hydroxylation of monohydroxyphenols to dihydroxyphenols and also causes

dehydrogenation of o-dihydroxyphenols to o-quinones (Mayer and Harel, 1979). So numbers

of factors are involved in PPO enzyme activity, among them moisture loss and humidity are

the major one (Akamine, 1960), as increase in PPO activity was observed when fruit were

stored at lower RH (Jiang and Fu, 1998). Some contradictory statements have been recorded

in the past regarding the activity of PPO. Activity of PPO enzyme was increased when stored


16

at 28oC and 76% RH within 48 h of harvest (Lin et al., 1988). On the other hand no

significant increase in the activity of PPO enzyme was observed after 48 h when fruit were

stored at 22oC and 85% RH (Zauberman et al., 1991). It was observed that activity of PPO

enzyme was dependent on RH during three days of storage. Litchi fruit stored at 90-95% RH

showed lower activity of PPO enzyme, as compared to 60-80% RH level showing higher

activity of PPO (Jiang and Fu, 1998). Underhill and Critchley (1993) gave contradictory

statement that activity of PPO was decreased at ambient conditions within 24 h of harvest.

Conflict among scientists in measuring the activity of PPO may be due to differences in

water contents of the fruit during harvesting, cultivar difference and different growing

conditions. Activity of PPO changes in browning susceptible and browning resistant cultivars

as activity of PPO enzyme was higher in browning susceptible litchi cv. ‘Nuomici’, than

browning resistant cv. ‘Guiwei’ (Chen et al., 2001). The presence of PPO enzyme activity

within the pericarp shows that PPO enzyme plays a significant role in desiccation browning.

Therefore, brown tissues of pericarp indicate that PPO enzyme activity is more pronounced

in epicarp and mesocarp tissues; whereas, endocarp tissues exhibit minor activity of PPO

enzyme. Percicarp protuberances from where browning initiates also show strong activity of

PPO enzyme (Underhill and Critchley, 1995). Activity of PPO enzyme tends to increase on

phenolic substrates, as PPO enzyme rapidly degraded anthocyanin contents in a solution

containing litchi phenolic extract, whereas, activity of PPO enzyme was minimum in a

solution without phenolic extract. Therefore, higher PPO enzyme activity in brown pericarp

tissues suggest that PPO enzyme play active role in regulation of desiccation browning in

litchi fruit (Jiang, 2000).

2.5.3.3 Peroxidase (POD)

Peroxidase (POD) in many fruit has been shown to cause enzymatic browning. It

involves in the breakdown of tissue and leads to senescence of the fruit. Sometimes, it

become difficult to identify between the two enzymes responsible for browning, as both POD

and PPO enzymes have similar action in causing browning. POD enzyme in the presence of

hydrogen peroxide acts on o-dihydroxyphenols to cause similar browning like PPO enzyme

(Kahn, 1985). The significance of POD enzyme in causing litchi pericarp browning is not

clear, as some scientists suggest it has major role in pericarp browning and some researchers


17

suggest it has minor role in browning (Underhill and Critchley, 1995; Jiang and Fu, 1998).

Activity of POD enzyme increased during early stages of pericarp browning during storage

but later on declined as storage period progressed (Lin et al., 1988; Jiang and Fu, 1998;

Huang et al., 1990). Storage conditions with relatively low RH favour activity of POD

enzyme and cause pericarp browning. Similarly, activity of POD enzyme is assumed to be

lower at higher RH conditions (Jiang and Fu, 1998). It seems that difference in opinion

regarding the activity of POD enzyme and its role depends on cultivar differences under

different storage conditions. In litchi cv. ‘Tai So’, lower activity of POD enzyme was

observed in epicarp than another litchi cv. ‘KMP’, which had higher activity of POD

enzyme; suggesting the role of POD enzyme varies considerably among different cultivars

(Underhill and Critchley, 1995).

2.5.3.4 Superoxide dismutase (SOD)

Fruit naturally contain SOD enzyme as a defense system against ROS (Niranjana et

al., 2009). SOD enzyme play major role in antioxidant defense system by maintaining the

ROS production below the threshold levels (Rao et al., 1996). At 20°C, the activity of SOD

in pulp tissues increased rapidly and began to decline 5 days later as fruit deteriorated (Chen

et al., 1987). However, Lin et al. (1988) found that the activities of SOD in peel and pulp

tissues of litchi fruit decreased gradually as the time of storage was extended and

malondialdehyde increased gradually at the same time. It showed that during the course of

storage, the ability of eradicating free radicals in the cells of both the pericarp and pulp

decreased gradually.

2.5.3.5 Pigments

Litchi fruit looks attractive and appealing due to its red colour, which is primarily due

the presence of anthocyanins (Lee and Wicker, 1991). The red, blue and purple colours of

plants are due to pigments known as anthocyanin (Sivakumar et al., 2007). These water

soluble pigments are situated in vacuole in the mesocarp and pericarp of litchi peel

(Underhill and Critchley, 1994). The anthocyanin contents become dense at fruit maturity in

mesocarp of the peel which later on decreased after harvest with increased activities of PPO

and POD enzymes (Markakis and Skoog, 1982). It is believed that PPO degrades

anthocyanin pigments and products of PPO oxidation are involved in browning of the litchi


18

pericarp (Lin et al., 1988; Tongdee, 1998). It was reported that after harvest as activity of

PPO enzyme increased in the litchi fruit, anthocyanin contents decreased resulting in pericarp

browning (Lin et al., 1988). Anthocyanin degradation is also related to moisture loss. At

ambient conditions (at 25°C and 60% RH) due to low humidity pericarp rapidly turned

brown with increased moisture loss and pericarp completely turned brown at 27%

degradation of total anthocyanins within 48 h (Underhill and Critchley, 1993). At slightly

higher humidity conditions (27-30°C and 73-79% RH), degradation of anthocyanin contents

was slower as only 10% anthocyanins were degraded after seven days of storage (Lin et al.,

1988). Naturally anthocyanin contents of litchi pericarp are soluble in water, but when

pericarp browning takes place, these red anthocyanin contents turn into brown pigments

which are only soluble in organic solvents such as acidic methanol (Song et al., 1996).

2.6 Management of pericarp browning

Different strategies have been reported to control the pericarp browning of litchi fruit.

Sulfur dioxide fumigation and acid treatments were suggested for pericarp colour retention

but sulfur dioxide fumigation may result in pericarp bleaching due the formation of

colourless anthocyanin complex (Holcroft and Mitcham, 1996). Sulphur dioxide fumigation

is commercially not acceptable because fruit absorb approximately 30-65% sulfur dioxide

which is beyond the maximum residual limit (10 ppm) allowed for sulfur in Europe,

Australia and Japan. In USA sulfur is register only for postharvest use on grape (Paull et al.,

1995). However, sulphur dioxide fumigation had been commercially applied to the litchi to

overcome postharvest browning but fumigation has certain demerits related to toxicity of its

residual level (Sivakumar et al., 2005).

2.6.1 Pre-cooling

Litchi fruit is harvested in June-July when the ambient temperature remains very high.

Therefore, litchi fruit require immediate protection against the high temperature in summer

season. To protect fruit from desiccation, pre-cooling is done to avoid pericarp browning,

extending shelf and storage life. Before transporting to any place fruit should be placed under

shade. Pre-cooling temperature is very important in reducing fruit weight loss. Pre-cooling

can be done in field, shed, storage at relatively low temperature after harvesting, sorting and

grading at relatively low temperature. It is very beneficial in preventing moisture loss by


19

reducing the temperature difference between fruit and air. Pre-cooling is helpful in avoiding

the problem of condensation which is caused due to temperature difference when fruit are

placed directly from warm to low temperature storage. A droplet of water is formed on the

fruit surface that reduces visual appearance, increase moisture loss and risk of rot

development. Similarly, if harvested fruit are packed without proper pre-cooling, then droplet

of water is formed on the surface of fruit which later on affects visual appearance (Joyce and

Patterson, 1994). Different techniques used for pre-cooling include dipping in cold water,

cold water sprays and using ice pellets or slurries. These techniques are very effective in

removing the field heat rapidly and conserve moisture content of the fruit. But the main

drawback of hydro-cooling is that water or moisture contents left at the fruit surface causes

pathogen attack and diseases during storage. It was observed that, hydro-cooling (0-1oC

water) took just 15 min to bring the temperature of litchi fruit from 27 to 3oC

(Pornchaloempong et al., 1997). Although, hydro-cooling was comparatively better in

retaining the fresh red colour of litchi pericarp but the risk of disease development is more, as

compared to fruit pre-cooled with air (Olesen and Wiltshire, 2000). Another technique like

forced air cooling can also be used to pre-cool the fruit. In this method cool air is forced

through the pile of fruit and it has shown benefits over simple low temperature room. Air

passes through the tiny holes and around the surface of fruit and the rate of cooling is much

quicker. As speed of cooling is much more therefore low RH air can be used to pre-cool the

fruit without excessive moisture loss (Joyce and Patterson, 1994). Therefore, forced air

cooling can be used in litchi as speed of cooling is high and also fruit does not get wet,

minimizing the danger of pathogen attack.

2.6.2 Heat treatment

Like other treatments, hot water treatment has also been established its significance in

improving quality of sweet pepper and mangoes (Fallik et al., 1999; Prusky et al., 1999). Hot

water treatments removes dirt from peel which contains fungal spores and it also stops

pathogen development (Shirra et al., 2000). Hot water brushing has been reported to inhibit

the activities of PPO enzyme by the pH of pericarp (Table 2.1). It allows uniform exposure of

pericarp to acid dipping and maintains the red form of anthocyanin pigments (Lichter et al.,

2000). Previously, hot benomyl treatment (48-50oC) for 2 min reduced pericarp browning


20

and maintained the red colour of ‘Kwai Mai Pink’ litchi pericarp (Wong et al., 1991).

Pericarp browning of two litchi cvs. ‘Tai So and ‘Wai Chee’ was delayed for 35 days under

cold storage when provided with vapour heat treatment at 45oC for 42 min (Jacobi et al.,

1993). Similarly, hot water brushing at 25oC for 20 sec followed by HCl dipping combined

with antifungal treatment with prochloraz improved eating quality and maintained red colour

of ‘Mauritius’ litchi pericarp for upto 35 days (Lichter et al., 2000). Hot water spray

treatment can also be effective in controlling pericarp browning. Fruit treated at 52oC for 2

min exhibited delayed browning and better quality than control fruit (Olesen et al., 2004). In

another study, fruit dipped in hot water (98oC) followed by treatment with oxalic acid

maintained higher anthocyanin level and reduced the activities of PPO and POD (Saengnil et

al., 2006). On contrary, hot water treatment depend can also result in quality loss of litchi

fruit (Follett and Sanxter, 2003). Hot water treatment at 50oC for 2 min had a harmful effect

on pericarp colour and deteriorated the peel structure of ‘McLean’s Red’ litchi fruit

(Sivakumar and Korsten, 2006).

Chapter‐2 Review of Literature

21

Table 2.1 Effect of heat treatment on pericarp browning and quality of litchi fruit

Treatment Cultivar Temperature/Dose Inference References

Hot benomyl ‘Kwai Mai Pink’ 48-50oC for 2 min Controlled pericarp browning with minimum loss in red colour

(Wong et al., 1991)

Heat treatment ‘McLean's Red’ 50oC for 2 min Caused pericarp browning and deteriorated pericarp structure

(Sivakumar and Korsten, 2006)

Hot water + oxalic acid

‘Hong Huay’ 98oC for 30 sec Inhibited browning by reducing the activities of PPO and POD enzymes

(Saengnil et al., 2006)

Hot water brushing + HCl dip + Prochloraz

‘Mauritius’ 25oC for 20 sec Maintained uniform red colour and good eating quality for 35 days

(Lichter et al., 2000)

Hot water spray 52oC for 2 min Delayed pericarp browning (Olesen et al., 2004)

Hot water dipping + irradiation treatment

‘Kaimana’ 49oC for 20 min + 400 Gy

Pericarp browning and quality loss was more in fruit treated with hot water as compared to irradiation treatment

(Follett and Sanxter, 2003)

Vapour heat treatment

‘Tai So’ and ‘Wai Chee’

45oC for 42 min Delayed pericarp browning for 4 weeks at 5oC

(Jacobi et al., 1993)

PPO = polyphenol oxidase, POD = peroxidase, HCl = hydrochloric acid


22

2.6.3 Irradiation

Irradiation treatment can be recommended as an alternative to SO2 for short term

storage under cold storage conditions (Table 2.2). Irradiation treatment upto 2 KGy inhibited

microbial growth on ‘Brewster’ litchi, and eliminated the chance of decay at 5 KGy (Kramer

and Kuhn, 1964). Similarly, irradiation treatment up to 1 KGy improved the visual

appearance and increased the market value of the litchi cv. ‘Thai’ for short period

(Ilangantileke et al., 1993). Storage life of ‘Tai So’ litchi was increased when fruit were

exposed to 1 KGy irradiation treatment followed by storage at 5oC (McLauchlan et al.,

1992). Lower dose of irradiation (0.5 KGy) increased the postharvest life of ‘Shahi’ and

‘China’ upto 28 days when under low temperature storage but could not significantly

improve the quality of fruit (Hajare et al., 2010). Gamma radiation treatment followed by

dipping in different solutions such as sodium hypochlorite, potassium metabisulphite,

ascorbic acid and HCl increased the storage life of ‘Shahi’ litchi upto 45 days (Kumar et al.,

2012). However, there are concerns among consumer about the safety of radiated food;

therefore, irradiation is not practiced commercially in many countries (Jiang et al., 2003).

2.6.4 Storage environment

2.6.4.1 Cold storage

Normally, freshly harvested litchi can be stored upto 20 days at 5°C, as pericarp

browning was less in fruit stored at 5°C than ambient temperature (Jiang and Chen, 1995).

However, litchi fruit deteriorates rapidly after removing from cold storage (Jiang and Li,

2003). Change in storage temperature also affects pericarp browning as fruit stored at 5°C

had better pericarp colour than fruit stored at 10 or 0°C (Mitra et al., 1996). Similarly fruit

stored at 5°C performed better and maintained superior colour than fruit stored at 2 or 0°C

(Huang and Wang, 1989). In another study, fruit stored at 1oC turned brown slowly than at

5°C but chilling injury problem appeared in fruit stored at 1°C (Zhang and Quantick, 2000).

Therefore, Olesen and Wiltshire (2000) optimized the storage temperature of litchi as 5°C.

But even under high humidity conditions, pericarp eventually turns brown after four weeks

due to cellular breakdown as a result of senescence (Underhill et al., 1992).


23

Table 2.2 Effect of irradiation on pericarp browning and quality of litchi fruit Treatment Cultivar Dose Inference References Irradiation ‘Brewster’ 2 KGy and

5 KGy Decay was completely controlled at 5 KGy irradiation treatment

(Kramer and Kuhn, 1964)

2 KGy Increased pericarp browning and deteriorated eating quality

(Akamine and Goo, 1977)

‘Tai So’ 1 KGy Reduced decay with extended storage life

(McLauchlan et al., 1992)

‘Thai’ 1KGy Maintained red colour and reduced fruit rot

(Ilangantileke et al., 1993)

‘Shahi’ and ‘China’

0.5 KGy At 4oC, increased postharvest storage life for 28 days

(Hajare et al., 2010)

‘Shahi’ and ‘China’

0.5 KGy Increased the cold storage life (4oC) of ‘Shahi’ and ‘China’ upto 45 and 30 days, respectively

(Kumar et al., 2012)

PPO = polyphenol oxidase, POD = peroxidase, KGy = Kilogray


24

However, it is necessary to store litchi at low but non-freezing temperature to delay

browning as litchi fruit quickly turns brown when at temperature above 10°C. Hence, it can

be said that optimum for litchi storage to retain red colour of pericarp appears to be between

1 to 5°C.

2.6.4.2 Controlled atmosphere storage

Postharvest storage life of litchi fruit can be enhanced by controlled atmosphere (CA)

storage (Table 2.3). CA storage with 3-5% O2 and 3-5% CO2% significantly increase the

storage life of fruit and delayed pericarp browning by inhibiting activities of PPO enzyme

(Jiang and Fu, 1999; Mahajan and Goswami, 2004). CA storage significantly reduced

pericarp browning for 6 days at 28ºC, under 100% oxygen (O2) and 0% carbon dioxide (CO2)

conditions by inhibiting the PPO enzyme. It is clear from their findings that activities of PPO

enzyme and anthocyanin contents being prime causes of pericarp browning can be inhibited

by pure oxygen O2 (Duan et al., 2004). In CA storage higher concentration of O2 maintained

good quality of litchi fruit by preventing microbial decay, limiting the activities of POD or

PPO enzymes, retaining higher concentration of anthocyanin contents and preventing of early

browning (Tian et al., 2005). 1-MCP treatment (500 nL/L) in combination with CA

conditions (3% O2 and 7% CO2) at 2°C for 21 days resulted in less pericarp browning with

low activities of oxidative enzymes, maintained high anthocyanin contents, delayed

senescence, conserve flavour and enhanced the overall acceptability of litchi fruit (Sivakumar

and Korsten, 2010).

2.6.4.3 Modified atmosphere packaging

Postharvest quality of litchi can be maintained by modified atmosphere packaging

(MAP) (Table 2.3). However, comprehensive effects of MAP on biochemical and

physiological alteration of litchi fruit have yet not been completely defined or clarified

(Somboonkaew and Terry, 2010). There are three major benefits of MAP i.e. it prevents

cross contamination, maintain high relative humidity, helps to prevent browning while

transportation and storage (Sivakumar et al., 2007). In MAP conditions, litchi fruit quality

remains fresh and storage life can be increased by using different range of storage

temperatures. Mangaraj et al. (2012) reported that fruit treated with ethylene

diaminetetraacetic acid (EDTA) and placed in MAP resulted in less browning, good taste and


25

extended storage life. On the other hand, litchi fruit packed in micro-perforated polyethylene

bags resulted in less fruit weight loss and decay, as compared to the non-microperforated

polyethylene bags (Chen et al., 2001). Somboonkaew and Terry (2010) reported that

packaging films were suitable to retain better quality of imported litchi cv. Mauritius fruit

during 9 days of storage, as compared to unwrapped fruit. PropaFreshTM PFAM is breathable

biaxially oriented polypropylene film with anti-mist properties significantly reduced sugar

transformation and retained anthocyanin contents resulted in brighter redness in the pericarp

as well as limited fruit weight loss, maintained anthocyanins, sugar, organic acids in both aril

and pericarp.

2.7 Role of different chemicals in pericarp browning

2.7.1 Oxalic acid

Oxalic acid is known as a natural antioxidant for natural and artificial preservation of

oxidized materials (Kayashima and Katayama, 2002). It has also shown properties of food

preservative and has gained attention as an anti-browning agent (Table 2.4) in different fruit

(Yoruk et al., 2002). Litchi cultivar ‘Huaizhi’ treated with 2 and 4 mM oxalic acid and stored

at room temperature significantly delayed the pericarp browning due to increase membrane

integrity, inhibition of anthocyanin breakdown, decline of oxidation and maintenance of

relatively low POD enzyme activity in the fruit during ambient storage (Zheng and Tian,

2006). Oxalic acid has also been reported to enhance the heat tolerance by increasing the

activity of POD and producing its isoforms (Zhang et al., 2001a). Anti-browning approaches

of oxalic acid for harvested litchi, longan and peach have been shown effective results

(Yoruk et al., 2004). It has been found that oxalic acid usage could be an effective

alternative to suppress postharvest deterioration and prolong the shelf-life of mango and

litchi fruit, linked with an initiation of higher activities of defense or anti-browning-

related enzymes such as POD and PPO (Zheng et al., 2011). Anti-browning approaches

of oxalic acid for harvested litchi, longan and peach have been shown effective results

(Yoruk et al., 2004). It has been found that oxalic acid usage could be an effective

alternative to suppress postharvest deterioration and prolong the shelf-life of mango and

litchi fruit, linked with an initiation of higher activities of defense or anti-browning-

related enzymes such as POD and PPO (Zheng et al., 2011).


26

Table 2.3 Effect of CA /MAP storage conditions on pericarp browning and quality of litchi fruit

Storage Cultivar Conditions Inference References CA storage ‘Heiye’ 70% O2 and 0% CO2 Limited ethanol production in aril (Tian et al., 2005) ‘Huaizhi’ 100% O2 and 0%

CO2 Reduced pericarp browning (Duan et al., 2004)

‘Huaizhi’ 3-5% O2 and 3-7.5% CO2

Increased storage life and reduced browning (Jiang and Fu, 1999) (Mahajan and Goswami, 2004)

CA + I-MCP

‘McLean's Red’ 3% O2 and 7% CO2 Reduced PPO and POD activities; maintained anthocyanin and delayed browning for 21 days

(Sivakumar and Korsten, 2010)

MAP ‘Mauritius’ Propafresh (PF) Maintained better quality (Somboonkaew and Terry, 2010)

‘Mauritius’ and ‘McLean's Red’

BOPP Decreased activities of PPO, POD enzymes and reduced pericarp browning

(De-Reuck et al., 2009)

‘McLean's Red’ BOPP Changed red colour to yellow and reduced PPO activity

(Sivakumar et al., 2008)

‘Shahi’ BOPP, PVC, PVDC + EDTA dipping (0.1%)

Maintained quality and delayed browning (Mangaraj et al., 2012)

BOPP = bi-axially oriented polypropylene, PVC = polyvinyl chloride, PVDC = polyvinylidene chloride, 1-MCP = 1-methylcyclopropene, POD = peroxidase, PPO = polyphenol oxidase.


27

Oxalic acid application increase SOD activity and maintained higher antioxidative

capacity of the banana fruit. Furthermore, potential of commercial storage of banana fruit at

room temperature became more pronounced when postharvest respiration, ethylene

production and ripening of banana fruit was inhibited by oxalic acid application (Huang et

al., 2013). At present no information is available on the combined application of oxalic acid

with other chemicals such as ascorbic acid and hexanal, which warrants further

investigations.

2.7.2 Ascorbic acid

Ascorbic acid has been used in litchi to control pericarp browning (Table 2.4). Major

role of ascorbic acid as a reducing agent in browning control is that it reduces quinone forms

of phenolics and phenyl radicals back to their precursors in oxidation reduction reaction.

Effects of ascorbic acid depend on its reducing activity and it can be used as a water soluble

antioxidant for preservation of food (Kitts, 1997). Ascorbic acid being a strong antioxidant

and antagonistic to ROS (Jimenez et al., 1997) has been reported to effectively control

enzymatic browning of fruits like apple and banana (Santerre et al., 1988; Rojas-Grau et al.,

2006). Chitosan combined with ascorbic acid plays active roles in inhibiting pericarp

browning, dehydration, microbial attack, maintaining membrane integrity and thus improve

litchi storability (Sun et al., 2010). At present to the best of my knowledge no information is

available on the postharvest exogenous application of ascorbic acid in combination with

oxalic acid and hexanal on the pericarp browning and quality of litchi fruit, which needs to

be investigated.

2.7.3 Hexanal

Hexanal is approved by the US Food and Drug Administration as a food additive and

it has an oral mammalian lethal dose (ORL-MAM LD50) of 3700 mg kg-1 (Song et al., 1996;

EAFUS, 2006). Hexanal as an aroma compound has been revealed as an anti-microbial agent

(Neri et al., 2006). Being a natural volatile compound with antimicrobial activity, hexanal

has been found to extend shelf life of the fruit and retain their original colour. In apple slices

its application increased the aromatic properties by metabolising different volatile

compounds (Song et al., 1996). It is strong inhibitor of POD enzyme activity, but methods of

hexanal application in fruits are under development (Paliyath and Subramanian, 2008). In


28

cherry fruit, it was observed that ethylene stimulated reactions could be inhibited by the use

of hexanal which improves nutritional quality, sensory evaluation and shelf life of cherry.

Hexanal vapors (450 ppm) applied for 48 h continuously reduced decay in apples which were

inoculated with P. expansum conidia. Hexanal applied in the same way like 1-MCP can

reduce decay in pear and apple (Sholberg and Randall, 2005). However, postharvest

application of hexanal has not been tested to control pericarp browning in litchi, which

warrants investigations.

2.7.4 Nitric oxide

Nitric oxide (NO) is a natural free radical gas, having multi-functional

signaling roles in in different plants (Wendehenne et al., 2001). Formerly, NO has

attracted great attention because of environmental pollutant but later on, it has been

revealed that it can control various physiological, pathological and developmental

progressions in plants (Lamattina et al., 2003; Neil et al., 2003). Furthermore, NO also

has additional evidences with anti-senescence and anti-ripening properties in several types of

fruits by decreased respiration rate, disease incidence, ethylene biosynthesis, delayed changes

in peel colour, and reduced enzymatic activities (Ku et al., 2000; Duan et al., 2007a;

Manjunatha et al., 2010). Recently, Barman et al. (2014) reported that 2 mM NO in the

form of sodium nitroprusside (SNP) decreased the pericarp browning, maintained higher

anthocyanin contents, phenolics, antioxidants, SSC, TA and ascorbic acid of litchi fruit

(Table 2.4).

2.7.5 Salicylic acid

Salicylic acid (SA) is one of the most important plant hormone in nature (Raskin,

1992). It is believed to have regulated several functions in the plant metabolism systems

(Popova et al., 1997). It has been found that SA being an endogenous plant growth regulator

from the phenolic group (Karlidag et al., 2009) used extensively for the quality improvement

in different types of fruit crops (Peng and Jiang, 2006). SA also significantly influence

different physiological and biochemical processes such as different fruit quality aspects,

membrane permeability, enzyme activities, growth and development (Arberg, 1981). Besides

different other fruit crops SA has also been reported to improve litchi fruit quality. SA

application at ambient conditions significantly reduced pericarp browning, activity of PPO


29

enzyme and ion leakage by maintaining higher anthocyanin contents, SSC and ascorbic acid

in litchi fruit (Kumar et al., 2013).

2.7.6 1-MCP

Even though litchi is a non-climacteric fruit in nature, treatment with 1-MCP

(0.25µL/L) has been reported to delay the activities of anthocyanase in ‘Guiwei’ litchi (Hu et

al., 2005). However, according to observations of Sivakumar et al. (2010), 1-MCP treatment

affects ethylene production, respiratory rate and activities of the PPO and POD enzymes.

Some other chemicals reported to reduce litchi pericarp browning are mentioned in table 2.4.

2.8 Browning in rambutan

The rambutan (Nephelium lappaceum L.) like litchi fruit has the aril as their edible,

fleshy part. It has hair like protuberance called spinterns which turn brown during postharvest

storage and result in major quality loss (Landrigan et al., 1996). It has been originated from

Malaysia and Sumatra but now the rambutan is produced commercially in Southeast Asia, as

well as in Sri Lanka, Australia, Central America, equatorial Africa and Malagasy (Delabarre,

1989). Browning in rambutan starts from sprinterns and then progresses towards the peel.

Moisture can be the major factor in Rambutan browning, as spinterns in rambutan have more

stomata and lenticels than peel, due to which transpiration takes place faster (Pantastico et

al., 1975). Following harvest, the visual appearance of rambutan can rapidly deteriorate

within 2-3 days if left under ambient conditions. Under low humidity conditions, rapid

moisture loss takes place through spintern due to which fruit loses it visual appearance and

quality is reduced (Landrigan et al., 1994). Like in litchi fruit, cold storage and heat

treatments have also been reported effective to control browning in rambutan. Therefore, it

can be said that browning in rambutan is probably influenced by the same factors which

promote pericarp browning in litchi (Lam et al., 1987). Postharvest storage life of rambutan

fruit was increased up to 16 days when stored at low temperature (8-10oC) and high humidity

conditions (Landrigan et al., 1996). The modified atmospheres and the use of plastic bags, as

well as card-board as wraps in poly propylene box have been used to extend the postharvest

life and reduce water loss (Paull et al., 1995). Browning in rambutan can be retarded by

application PPO and POD inhibitor like salicylhydroxamic acid and catalase (Landrigan et

al., 1996). In situ acidification with Lactobacilli plantarum, alone or in combination with


30

chitosan, reduced browning and maintained quality of rambutan fruit (Martínez-Castellanos

et al., 2009).

2.9 Browning in longan

Longan (Dimocarpus longan Lour.) is a non-climacteric fruit and does not ripe after

harvest (Paull and Chen, 1987). It can be grown in tropical areas (Nicholls, 2001), as it has

been originated from Northern Burma and China (Partridge, 1997). Commercially it is grown

in China, India, Thailand and Vietnam (Campbell and Campbell, 2001). It has very short

shelf life irrespective to cultivar and deteriorated within 3-4 days after harvest at ambient

conditions (Paull and Chen, 1987) due to which its consumption is limited. Pericarp

browning is the major factor which affects the storage life and quality of longan fruit

(Suwanakood et al., 2004). Pericarp browning is mainly due to enzymatic reaction of PPO

which greatly affects its market value (Jiang et al., 2002). Covering the fruit with

polyethylene bags or storage in plastics container delays pericarp browning and extends shelf

life of longan fruit. Storage temperature for longan depends upon cultivar; however, it can be

stored for 30 days at 1-5oC (Underhill et al., 1992). Sulfur dioxide treatments are widely used

to control saprophytic surface fungi and prevent peel browning of longan fruit (Han et al.,

1999). Similarly, controlled atmospheres (CA) storage significantly reduced decay, prevented

peel browning and extended storage life of longan fruit (Tian et al., 2001).


31

Table 2.4 Effect of exogenous application of different chemical on pericarp browning and quality of litchi fruit

Chemical Cultivar Dose Inference References

1-MCP ‘Guiwei’ Delayed anthocyanase activity (Hu et al., 2005)

‘McLean's Red’

500 µL L-

1 Reduced browning, red colour (colour value a*), ascorbic acid, PPO, POD enzyme activities.

(Sivakumar et al., 2010)

ACS ‘Brewster’ 2.5 or 5%

Delayed browning; reduced PPO and POD activities; increased total phenolics and antioxidants

(Wang et al., 2010)

Ascorbic acid ‘Feizixiao’ 40 mM Reduced the activities of POD and PPO enzymes when stored at 5oC

(Sun et al., 2010)

ATP

‘Huaizhi’ ‘Huaizhi’

1 mM 1 mM

Delayed pericarp browning Inhibiting pericarp browning and ROS accumulation

(Song et al., 2006) (Yang et al., 2009)

Chitosan

‘Feizixiao’

1% Inhibited pericarp browning, dehydration, microbial attack; maintained membrane integrity; extended storage life for 30 days

(Sun et al., 2010)

‘Huaizhi’

1% Delayed anthocyanin degradation; inhibited PPO activity; reduced pericarp browning

(Zhang and Quantick, 1997)

‘Huaizhi’ 2% Inhibited PPO activity; maintained higher anthocyanin contents; improved fruit quality for 12 h at ambient (25oC) conditions after removal from cold storage at 2oC

(Jiang et al., 2005)

‘Heli’ 1% Reduced respiration and moisture loss (Song et al., 2006) ‘Mauritius’ 1% Reduced microbial decay (Sivakumar et al., 2005) ‘Huaizhi’ 1 mM Inhibited ROS accumulation and pericarp browning (Yang et al., 2009)

1 MCP = 1-methylcyclopropene, ATP = adenosine triphosphate, ACS = acidified calcium sulfate, POD = peroxidase, PPO = polyphenol oxidase, ROS = reactive oxygen species


32

Table 2.4 Continued

Chemical Cultivar Dose Inference References

Glutathione + Citric acid

‘Huaizhi’ 10 mM and 100 mM

Effectively inhibited PPO activity and reduced pericarp browning

(Jiang and Fu, 1998)

HCl ‘Bengal’ 1 mM with PVC Controlled browning for 20 days (Silva et al., 2012)

‘Huaizhi’ 1% Delayed browning (Zauberman et al., 1991)

Oxalic acid ‘Huaizhi’ (2 or 4 mM) Delayed pericarp browning and anthocyanin breakdown; increased membrane integrity; reduced POD and PPO activity

(Zheng and Tian, 2006)

‘Hong Huay’ 10% Retained red colour (Saengnil et al., 2006)

NO ‘China’ 2 mM Decreased pericarp browning; increased anthocyanin contents, phenolics, antioxidants, SSC, TA and ascorbic acid

(Barman et al., 2014)

Polyamines ( PUT, SPD, SPM)

‘Huaizhi’ 1 mM SPM was most effective in delaying peroxidation, senescence, pericarp browning, membrane leakage when stored at 5oC for 30 days

(Jiang and Chen, 1995)

NO = nitric oxide, HCl = hydrochloric acid, PVC = polyvinyl chloride, PUT = putrescine, SPD = spermidine, SPM = spermine, POD = peroxidase, SSC = soluble solid concentrations, TA = titratable acidity, PPO = polyphenol oxidase.


33

2.10 Conclusion

The literature review explained the capability of current commercial practices to

restrict water loss and prevent pericarp browning. From above discussion it is also clear that

harvest locations, cultivars and postharvest application of some chemicals like ascorbic acid

and oxalic acid have the potential to control postharvest pericarp browning in litchi.

However, potential of hexanal to control postharvest browning in litchi is yet to be

investigated. Hence, present study was undertaken to investigate the effect of harvest

locations, cultivars, exogenous application of oxalic acid, ascorbic acid and hexanal on the

onset of litchi pericarp browning and other physical and biochemical attributes of litchi

during storage.

Chapter‐3 General materials and methods

34

Chapter 3

GENERAL MATERIALS AND METHODS

The present study was carried out in Postharvest research and training center (PRTC),

Institute of Horticultural Sciences, University of Agriculture Faisalabad. The materials and

methods used in the study are as follows:

3.1 Experimental material

3.1.1 Litchi fruit source

Commercial and well maintained orchards were selected from two different locations

i.e. Saggian Bridge (31o35.511’N, 74o15.375’E), Lahore district, Punjab and Fruit Farm

Nursery (34o00.114’N, 72o56.779’E), Haripur, KPK (Fig. 3.1).

Fig. 3.1 Harvest locations: Haripur, KPK (A) and Lahore, Punjab (B)

(Source: https://www.google.com/maps/@31.7829414,74.2077715,9z?hl=en-US)

(A)

(B)


35

Fruit of two litchi cvs. ‘Gola’ and ‘Serai’ (Fig. 3.2) were harvested from 30-35 year

old uniform and healthy trees planted at 20 m × 20 m apart (in rows and between plants) and

grown under uniform soil conditions. After harvest fruit were transported to Postharvest

Research and Training Center (PRTC) in reefer van at 12oC. Fruit of uniform, size, shape,

free from any physical damage were selected for the study and kept at ambient conditions

(25±2oC and 65±5% RH) for 5 days.

Fig. 3.2 Fruits of two litchi cultivars used for study.

3.1.2 Climate of harvest locations

According to Koppen and Geiger climate systemx, Lahore locations is classified as

BShy while Haripur as Cwaz [(x = Koppen–Geiger climate classification, y =Subtropical dry

semiarid type climate (Steppe), z = Humid subtropical climate] (Peel et al., 2007). The

warmest month of the year at Lahore and Haripur is June with an average temperature of

33.9°C and 32°C, respectively. Both locations share November as the driest month with 4

mm and 15 mm precipitations in Lahore and Haripur, respectively. Most precipitation falls in

August, with an average of 189 mm at Lahore, while, 175 mm at Haripur. The lowest

average temperature of the whole year falls in the month of January at both locations, the

average January temperature is 12.3°C at Lahore, while, at Haripur it is 10.1°C. The

difference in precipitation between the driest month and the wettest month at Lahore and

Litchi fruit cv. Serai Litchi fruit cv. Gola


36

Haripur is 185 mm and 160 mm, respectively. The other climatic characteristics are given in

table 3.1.

Table 3.1 Climatic characteristics of locations used for the study

Climatic characteristics Lahore, Punjab Haripur, KPK Geographic coordinates 74°15.3′75″ E;

31°35.5′11″ N 72°56.7′ 79″ E; 34°0.1′14″ N

Altitude 217 m 537 m

Annual temperature 24.2 °C 21.5 °C

Annual rainfall 607 mm 832 mm

Annual temperature variation 21.6 °C 21.9 °C

(Source: climate data.org; available at http://en.climate-data.org/location/3077/).

3.2 Storage conditions

Litchi fruit used in this integrated study were treated according to experimental

treatments (detail given in each experiment section). Treated and untreated litchi fruit were

stored at ambient (25±2oC and 65±5% RH) and under cold storage conditions (5±1oC and

90±5% RH).

3.3 Parameters studied

The data were collected for the following fruit quality parameters.

3.3.1 Physical fruit quality characteristics

3.3.1.1 Pericarp browning index

Extent of pericarp browning was assessed by measuring the extent of the total

browned area on each fruit pericarp, using the scale described by Jiang and Chen (1995) with

some modifications (Table 3.2). The browning index was calculated as browning scale % of

corresponding fruit within each class. Fruit lots at higher than 3.0 pericarp browning index

were considered unacceptable for marketing.


37

Table 3.2 Scale used for browning assessment

Pericarp browning index

1 = 0. no browning (excellent quality)

2 = 1. slight browning

3 = 2. < ¼ browning

4 = 3. ¼ to ½ browning

5 = 4. > ½ to ¾ browning (poor quality)

6 = 5. >3/4 browning (very poor quality)

The browning index was calculated using the formula:

∑ (browning scale x percentage of corresponding fruit within each class).

3.3.1.2 Fruit weight loss (%)

Fruit samples were weighed before and after storage with the help of digital balance

(MJ-W176P, Panasonic Japan). Weight loss % was calculated according to the formula as

given below

Weight loss (%)

3.3.2 Chemical characteristics

3.3.2.1 Soluble solid concentrations (SSC) (Brix°)

Litchi pulp tissues from 25 fruit were pureed in a juicer/blender (MJ-W176P,

Panasonic, Japan was used to determine SSC of litchi fruit. Before determining SSC, juice

was filtered through Whatman® filter paper. Digital Refractometer (RX 5000, Atago, Japan)

was used for the determination of soluble solid concentrations (SSC). A drop of juice was

placed on the prism of refractometer, and SSC (°Brix) was noted directly from the digital

scale of refractometer at room temperature.


38

3.3.2.2 Titratable acidity (TA) (%)

Titratable acidity (TA) of fruit juice was determined by method given by Hortwitz

(1960). Ten mL of juice was taken in 100 mL conical flask and diluted up to 50 mL with

distilled water. It was titrated against 0.1 N NaOH, using 2-3 drops of phenolphthalein as an

indicator till pink colour end point was achieved. To determine TA calculations were made

using milli-equivalent factor for malic acid, 0.067, the major acid in litchi at maturity (Huang

and Scott, 1985).

TA (%) =

3.3.2.3 SSC: TA ratio

SSC: TA was calculated in each sample by dividing the percentage of SSC with the

percentage of the TA.

3.3.2.4 Ascorbic acid (mg 100 g-1)

For the estimation of ascorbic acid in the pulp, the method described by Khalid et al.

(2012) was used. For this purpose extracted juice from each sample was filtered through

Whatman® filter paper. Ten mL of filtered aliquot was taken in 100 mL round bottom flask,

then volume was made up to the mark by adding 0.4% oxalic acid. Out of 100 mL aliquot, 5

mL was taken in a beaker and titrated against freshly prepared dye (2, 6-dichlorophenol

indophenol) till light pink end point appeared which persisted for 10-15 seconds.

Ascorbic acid (mg 100 g-1) =

Where

D1 = mL dye used in titration of aliquot

D = mL of dye used in titration of 1 mL standard ascorbic acid solution prepared by

adding 1 mL of 0.1% ascorbic acid + 1.5 mL of 0.4% oxalic acid

A = mL of juice used

V = volume of aliquot made by addition of 0.4% oxalic acid


39

B = mL of aliquot used for titration

Preparation of dye

Dye was prepared by adding 42 mg NaHCO3 and 52 mg 2, 6-dichlorophenol

indophenols in a 200 mL volumetric flask. Volume was made up to the mark by distilled

water, filtered and always freshly prepared dye was used.

3.4. Determination of total phenolics (TP) and total antioxidants

One g of peel or pulp sample (flash frozen with liquid nitrogen and stored at -80oC)

was taken and homogenize in mortar and pestle by adding 5 mL of methanol: acetone: HCL

solution (90:8:2). After homogenizing, one sample was put in two eppendorf tubes and

vortexed with the help of vortex mixer (SLV-6, MyLabTM, Seoulin BioScience, Korea) for 2

min. Samples were centrifuged (13000 × g for 3 min at room temperature) and supernatant

was collected in a fresh eppendorf tubes (Fig. 3.3).

Fig. 3.3 Flow chart for extraction of TP and total antioxidants in peel and pulp tissues of litchi fruit

Pellet (discarded) Supernatant (Total antioxidants and TP analysis)

Pulp/Peel sample (1 g)

Centrifugation (10,000 × g, 5 min, 4oC)

Homogenization

5 mL extraction solution (Methanol: acetone: HCl in 10: 8: 2 ratio + 200 mg sand)


40

Activity calculation (mg GAE 100 g-1)

Absorbance (765 nm)

Addition

200 µL supernatant + 200 µL FC-reagent

Vortex mixing (10 s)

Vortex mixing (10 s)

3.4.1 Total phenolics (TP)

The TP of litchi fruit was determined using the Folin–Ciocalteu (FC) method as

outlined by Ainsworth and Gillespie (2007). The extracted samples (100 µL) were mixed

with 200 µL FC-reagent in a micro centrifuge tube and vortexed thoroughly with the help of

vortex mixer (SLV-6, MyLabTM, Seoulin BioScience, Korea) for 1 min (Fig. 3.4). After

adding 800 µL of 700 mM sodium carbonate (Na2CO3), the tube was again vortexed for 1

min and incubated at room temperature for 2 h. The 200 µL mixture from each sample was

poured into a 96-well plate. Then plate was read at 765 nm by microplate reader (ELX800,

Bio-Tek Instruments, Inc.,Winooski, VT, USA). The TP were expressed as mg GAE 100 g-1

against the standard curve of gallic acid (Fig. 3.5).

Fig. 3.4 Flow chart for determination of TP in peel or pulp tissues of litchi fruit


41

R² = 0.9984

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 200 400 600 800 1000 1200

Ab

sorb

ance

un

its

mg/L Gallic acid

Fig. 3.5 Standard curve of gallic acid for determination of TP in peel or pulp tissues of litchi fruit

3.4.2 Total antioxidants

Total antioxidants in peel and pulp tissues of litchi fruit were determined by using

2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay as described by Brand-Williams et al.

(1995). The extracted supernatant (50 µL) of the pulp extract was added to 5 ml 0.004%

methanolic solution of DPPH in a glass test tube (Fig. 3.6). For total antioxidants

determination, different amounts of sample (50, 100, 150 µL) were tested with 30 min

interval. The test tubes were covered with aluminum foil and kept in dark conditions as well

to avoid any light effect. The decrease in absorbance was determined at 517 nm at an interval

of 30 min until the reaction reached a plateau. The absorbance was recorded by microplate

reader (ELX800, Bio-Tek Instruments, Inc.,Winooski, VT, USA). The final reading was

given as % inhibition of DPPH and calculated by the formula given below:

Where

A1 = absorbance of the blank sample (containing DPPH without sample)

A2 = absorbance of DPPH after adding the sample


42

Activity calculation (% scavenging DPPH)

Absorbance

50 µL supernatant + 5 mL 0.004% DPPH•

Incubation (30 min, 25°C, dark conditions)

Addition (50 µL supernatant)

Absorbance

Addition (50 µL supernatant)

Absorbance

Fig. 3.6 Flow chart for determination of total antioxidants in peel or pulp tissues of litchi fruit

3.5 Determination of anthocyanin contents

Pericarp from 25 fruit (flash frozen with liquid nitrogen and stored at -80oC) was

pooled and mixed with a mixer and blender (MJ-W176P Panasonic, Japan). A sample of one

g was taken in a falcon tube containing 10 mL extraction mixture HCl–methanol (15:85) as

per protocol of (Proctor, 1974). The sample was incubated at 25 ̊C for 1 h and finally it was

centrifuged at 4000 × g in a centrifuge machine (Rotofix 46 tabletop, Hettich, Germany)

(Fig. 3.7).


43

Fig. 3.7 Flow chart for extraction of anthocyanin contents in peel tissues of litchi fruit

The 200 µL supernatant was poured into a 96-wellplate. The ELX800 Microplate

Reader (Bio-Tek Instruments, Inc.,Winooski, VT, USA) was used to determine the

absorbance at 530, 620 and 650 nm, respectively. The anthocyanin content measurement was

calculated based on the formula as suggested by (Zheng and Tian, 2006). Anthocyanin

contents were expressed as change in absorbance per gram of peel fresh weight.

∆A g-1FW= (A530- A620) - 0.1 (A650- A620)

Where,

∆A = change in absorbance

A530 = absorbance at 530 nm



3.6 Determination of activities of anti-oxidative enzymes

Litchi peel and pulp tissue were flashed frozen in liquid nitrogen and stored at -80oC

until utilization. Frozen samples (1 g) were homogenized in 2 mL potassium phosphate

buffer (7.2 pH) using pre-chilled mortar and pestle on ice plate followed by centrifugation at

10,000 × g for 5 min at 4°C. The supernatant was collected and used for determination of

antioxidative enzymes (Fig. 3.8).

Pellet (discarded) Supernatant (anthocyanin contents)

Peel sample (1 g)


Homogenization

10 mL extraction solution (Methanol: HCl in 85: 15)


44

Fig. 3.8 Flow chart for extraction of antioxidative enzymes in peel or pulp tissues of litchi

fruit

Peel and pulp tissue of litchi fruit were flashed frozen in liquid nitrogen and stored at

-80oC until utilization. Frozen samples (1 g) were homogenized in 2 mL potassium citrate

buffer (4.0 pH) containing polyvinylpyrollidon (PVP) and then centrifuged at 10,000 × g for

5 min at 4°C. The supernatant was collected and used for determination of PPO enzyme

activity (Fig. 3.9).

Fig. 3.9 Flow chart for extraction of polyphenol oxidase enzyme in peel tissues of litchi fruit

Pellet (Discarded) Supernatant (PPO analysis)

Peel tissue sample (1 g)


Homogenization

2 mL potassium citrate buffer + PVP

Pellet (Discarded) Supernatant (CAT, POD and SOD analysis)

Peel/Pulp tissue sample (1 g)


Homogenization

2 mL potassium phosphate buffer


45

3.6.1 Catalase (CAT)

CAT activity in litchi peel and pulp samples were determined by using method as

outlined by Liu et al. (2009) with some modifications. Enzyme extract (100 µL) was mixed

with freshly prepared 5.9 mM 100 µL H2O2 to initiate enzyme reaction. CAT activity

was determined by ELX800 Microplate Reader (Bio-Tek Instruments, Inc.,Winooski, VT,

USA) at 240 nm and was expressed as U mg−1 protein (Fig. 3.10). One unit of CAT activity

was defined as “an absorbance change in 0.01 units per min”.

Fig. 3.10 Flow chart for determination of activity of CAT enzymes in peel or pulp tissues of

litchi fruit

3.6.2 Peroxidase (POD)

POD activity was analyzed using the method of Liu et al. (2009) with some

modification. Fresh reaction mixture was prepared by adding 50 mM 800 µL phosphate

buffer (5.0 pH) into 40 mM 100 µL H2O2 and 20 mM 100 µL guaiacol. Absorbance of

enzyme extract (100 µL) mixed with reaction mixtures (100 µL) was recorded at 470 nm

using ELX800 Microplate Reader (Bio-Tek Instruments, Inc.,Winooski, VT, USA) and was

expressed as U mg−1 protein (Fig. 3.11). One unit of POD activity was defined as “an

absorbance change in 0.01 units per min”.

Activity calculation (U mg-1 protein)

100 µL supernatant

100 µL H2O2 5.9 mM, (35% pure)

Absorbance (240 nm)


46

Fig. 3.11 Flow chart for determination of activities of POD enzymes in peel or pulp tissues of litchi fruit

3.6.3 Superoxide dismutase (SOD)

SOD assay was carried out by measuring the 50% inhibition of the photochemical

reduction of nitro blue tetrazolium (NBT) according to the method described by Stagner and

Popovic (2009). Each test tube contained 500 µL phosphate buffer (50 mM, 5.0 pH),

methionine 200 µL (22 µM), 100 µL NBT (20 µM), 200 µL Triton X (0.1 µM), 100 µL

riboflavin (0.6 µM) as a substrate and 800 µL distilled water was mixed with 100 µL enzyme

extract. Test tubes were kept in a box illuminated with UV-fluorescent lamps for 15 min. The

absorbance was recorded at 560 nm by using ELX800 Microplate Reader (Bio-Tek

Instruments, Inc.,Winooski, VT, USA) and was expressed as U mg−1 protein (Fig. 3.12). One

unit of SOD activity was defined as “the amount of enzyme that inhibited 50% of NBT

photo-reduction”.


100 µL supernatant

100 µL reaction mixture (0.1 M Potassium Phosphate buffer, 5 pH, 40 mM H2O2: 20 mM

guaiacol in 8: 1: 1)

Absorbance (470 nm)


47

100 µL 20 µM nitro-blue tetrazolium

200 µL 0.1 µM Triton X

UV-fluorescent light (400 nm) (15 min)


100 µL supernatant

500 µL potassium phosphate buffer (50 mM, 5 pH)

200 µL 22 µM methionine

800 µL distilled water

Absorbance

Fig. 3.12 Flow chart for determination of activity of SOD enzymes in peel or pulp tissues of litchi fruit

3.6.4 Polyphenol oxidase (PPO)

The assay mixture for PPO contained supernatant 50 µL, freshly prepared 1.45 mL

100 mM potassium citrate buffer (6.8 pH) and 0.50 mL 100 mM 4-methylcatechol (4-MC).

The absorption was noted at 412 nm on ELX800 Microplate Reader (Bio-Tek Instruments,

Inc.,Winooski, VT, USA) (Fig. 3.13). PPO activity was determined as enzyme units (U mg-1

protein), defined as the quantity of enzyme required to produce 1 µmoL min-1 product

(Waite, 1976).


48

Fig. 3.13 Flow chart for determination of activity of PPO enzyme in peel tissues of litchi fruit

3.7 Statistical Analysis:

The experimental data were subjected to analysis of variance (ANOVA) using

Statistix 10 for Windows software with three-factor factorial arrangements including

cultivars, treatments and fruit storage period. Each experimental unit was consisted of 25

fruits with three replicates. The effects of treatments were determined from the least

significant differences test (Fisher’s LSD) at P ≤ 0.05, where the F test was significant (Steel

et al., 1997). Pearson’s correlations were also performed to estimate relationship between

fruit browning index, anthocyanin contents and activities various antioxidative enzyme using

Statistix 10 (Analytical Software, Tallahasee, FL 32317, USA).


50 µL supernatant + 1.45 mL potassium citrate buffer + 0.5 mL 4-MC (100 mM)

Absorbance (412 nm)

Chapter‐4 Locations and cultivars affect quality of litchi fruit

49

Chapter 4

Harvest Locations and Cultivars Influence Pericarp Browning and Biochemical Quality of Litchi Fruit

4.1 Abstract

Percicarp browning limits the postharvest life of the litchi fruit with reduced fruit

quality. Therefore, present study was conducted to investigate the influence of harvest

locations (Lahore and Haripur) and cultivars (‘Gola’ and ‘Serai’) on pericarp browning,

anthocyanin contents and changes in the biochemical quality attributes [soluble solid

concentrations (SSC), titratable acidity (TA), SSC: TA ratio, ascorbic acid] were determined

in pulp tissues, whereas, total phenolics (TP), total antioxidants and enzymatic activities

[superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase

(PPO)] were determined in litchi peel and pulp tissues. Fruit harvested from Lahore exhibited

higher pericarp browning index, weight loss, ascorbic acid, and activities of POD and PPO

enzymes, as compared to Haripur. Moreover, fruit harvested from Lahore exhibited less SSC:

TA ratio, TP, total antioxidants and activity of SOD enzyme than Haripur. On the other hand,

‘Serai’ fruit exhibited higher fruit weight loss, SSC, TA, activities of POD and PPO enzymes

with lower level of TP, total antioxidants, activities of SOD and CAT enzymes than ‘Gola’

fruit. Results suggested that fruit harvested from Haripur exhibited better fruit quality

characteristics than Lahore with less browning index and ‘Gola’ cultivar performed better

than ‘Serai’.

4.2 Introduction

Litchi (Litchi chinensis Sonn.) belongs to tropical and subtropical areas that

originated near Southern China and Northern Vietnam. Litchi is an important commercial

fruit crop in tropical and sub-tropical areas of the world with China being the largest

producer (Menzel, 2001). In Pakistan, litchi is grown on an area of about 572 ha with 9250

tons production (Shah, 2003). It is highly nutritious and can meet 2-4% dietary recommended

intake of most of minerals. Consumption of 14-15 litchis per day covers up daily average

vitamin C requirement (Wall, 2006). However, mineral contents of litchi fruit differ between


50

cultivars even though harvested from same locations (Shewfelt, 1990). Due to high

nutritional value, the demand of litchi is increasing day by day in Pakistan.

Postharvest pericarp browning is the main issue in litchi fruit which involves rapid

browning of fruit skin after harvest (Kumar et al., 2013), thus affecting the fruit quality and

hindering its market potential (Rajwana et al., 2010). Different biochemical and

physiological changes and enzyme analysis such as, reduction of membrane integrity,

degradation of anthocyanins, increase in peroxidase (POD), polyphenol oxidase (PPO)

activities, and catalyzation of superoxidation reaction have been reported to be associated

with pericarp browning in litchi (Jiang, 2000). Several efforts have been made to overcome

the pericarp browning issue by using postharvest fruit coatings (Joas et al., 2005), different

antioxidants (Kumar et al., 2013), oxalic acid (Zheng and Tian, 2006) and ascorbic acid (Sun

et al., 2010), however, with variable and sporadic results. These variable results could be due

to season, location and cultivar differences (Luton and Holland, 1986; Knee and Smith, 1989;

Guerra and Casquero, 2009).

At present no information is available about the effect of harvest locations and

cultivars on the pericarp browning and fruit quality of litchi. We hypothesize that incidence

of pericarp browning and quality will be affected by locations of harvest and cultivars.

Therefore, it is the need of the time to further investigate that which area and cultivar is best

suited for litchi cultivation with reduced pericarp browning at ambient conditions. Hence,

present study was conducted to determine the effect of locations and cultivars on pericarp

browning, changes in enzymatic activity and fruit quality of litchi at ambient conditions.

4.3 Materials and Methods

Plant material

Fruit of litchi (Litchi chinensis Sonn.) cv. ‘Gola and ‘Serai’ was harvested at

commercial maturity (SSC = 17.5oBrix, TA = 0.4%) from two locations i.e. Saggian Bridge

Lahore (31o35.511’N, 74o15.375’E) and Fruit Farm Nursery Haripur (34o00.114’N,

72o56.779’E). After harvest fruits were transported to Postharvest Research and Training

Center (PRTC) in reefer van as described in detail in section 3.1.1 (Page 34). The data were


51

recorded regarding fruit weight loss (%) and pericarp browning index. Fruit quality

parameters [soluble solid concentrations (SSC), titratable acidity (TA), SSC: TA ratio,

ascorbic acid] were determined in pulp tissues, whereas, total phenolics (TP), total

antioxidants, anthocyanins contents and activities of peroxidase (POD), polyphenol oxidase

(PPO), catalase (CAT) and superoxide dismutase (SOD) enzymes were determined in peel

and pulp tissues of litchi fruit.

Pericarp browning

Pericarp browning was determined by using the method described in detail in section

3.3.1.1 (Page 36). Pericarp browning was expressed as browning index.

Fruit weight loss

Fruit weight loss was calculated by using digital balance as described detail in section

3.3.1.2 (Page 37) and was given in percentage of fruit weight loss.

Biochemical analysis

SSC (oBrix) of fruit juice was determined with a digital refractometer (Section

3.3.2.1; page 37). Titratable acidity (TA) was determined by titration method as described in

detail in section 3.3.2.2 (Page 38) and was expressed in percentage of malic acid.

Anthocyanin contents

Anthocyanin contents in peel tissues of litchi fruit were determined by using method

outlined in section 3.5 (Page 42) and was expressed as ∆A g-1FW.

Total phenolics, total antioxidants and ascorbic acid

Total phenolics (TP) from litchi peel and pulp tissues were determined by the method

described in detail in section 3.4.1 (Page 40). The concentration of TP was expressed as the

gallic acid equivalent (GAE mg g-1). The DPPH free radical scavenging activity of total

antioxidants were measured in peel and pulp tissues of litchi fruit by using method outlined

in detail in section 3.4.2 (Page 41) and were expressed as percentage inhibition of DPPH

radical. Determination of ascorbic acid was carried out according to method outlined in

section 3.3.2.4 (Page 38) and was expressed in mg 100 g-1.


52


For determination of antioxidative enzyme (CAT, SOD, POD, PPO), the supernatant

was extracted according to the method outlined in section 3.6 (Page 43). CAT enzyme was

measured as U mg-1 protein (section 3.6.1; page 45), where one unit was defined as “an

absorbance change in 0.01 unit min-1”. POD enzyme activity was determined by the guaiacol

method (section 3.6.2; page 45). SOD enzyme activity was measured in terms of its capacity

to inhibit photochemical reduction of nitroblue tetrazolium (NBT) according to method

described in section 3.6.3 (Page 46). PPO enzyme activity was determined as enzyme units

(U mg-1 protein), defined as the quantity of enzyme required to produce 1 µmoL min-1

product (section 3.6.4; page 47).

Statistical analysis


Statistix 10 for Windows software with three-factor factorial arrangements including

cultivars, harvest locations and fruit shelf period. Each experimental unit was consisted of 25

fruits with three replicates. The effects of treatments were determined from the least


et al., 1997).

4.4 Results

Fruit weight loss

Fruit weight loss showed a significant increasing trend with increase in shelf period

regardless of cultivars and harvest locations. However, highest fruit weight loss (1.05-fold)

was exhibited by ‘Serai’ fruit harvested from Lahore on day-5 of shelf period (Table 4.1).

Overall fruit of litchi cultivar ‘Gola’ exhibited 1.14-fold less weight loss than ‘Serai’ (Table

4.2).

Pericarp browning, PPO activity and Anthocyanin

Pericarp browning index was significantly and gradually increased with progress in

shelf period (day-1 to day-5), irrespective to locations of harvest and cultivars (Fig. 4.1A and

D). On day-5 of shelf period fruit harvested from Lahore showed 1.6-fold higher pericarp


53

browning index than Haripur. However, no significant difference among cultivars was

noticed regarding their browning index (Table 4.2). Increasing trend was observed in the

activity of PPO enzyme from Day-1 to Day-5 of shelf period (Fig. 4.1C and F). Overall fruit

of litchi cv. ‘Gola’ showed significantly about 1.1-fold less PPO enzyme activity than

‘Serai’. Fruit harvested from Lahore exhibited 1.20-fold higher activity of PPO enzyme than

from Haripur (Table 4.2). Overall decreasing trend in anthocyanin concentration was

observed with increase in shelf life period. Non-significant increase in anthocyanin

concentration was observed on day-3 but a significant decline was noticed at day-5 of shelf

period (Fig. 4.1B and E). Non-significant differences were observed between locations of

harvest and cultivars (Table 4.2).

SSC, TA, SSC: TA and Ascorbic acid

Significant increase in SSC of fruit juice was observed as number of days at shelf

period progressed. SSC showed increasing trend in both cultivars (‘Gola’ and ‘Serai’),

irrespective of harvest locations. It was observed that ‘Serai’ fruit exhibited higher SSC, as

compared to ‘Gola’ in both locations with increase in shelf life period (Table 4.1). There was

no significant difference in SSC with respect to locations. ‘Serai’ fruit exhibited 1.2-fold

higher SSC, as compared to ‘Gola’ fruit (Table 4.2). The titratable acidity (TA) percentage

showed decreasing trend with increase in number of days at shelf period with maximum TA

calculated on day-1 of shelf period (0.33%) and minimum TA was recorded on day-5

(0.10%) (Table 4.1). Significant increase in SSC: TA ratio was noticed as shelf period

increased with minimum SSC: TA being 46.2% on day-1 of shelf period in fruit of ‘Gola’

cultivar harvested from Lahore and maximum SSC: TA was noted on day-5 of shelf period

(482.6%) (Table 4.1). Fruit harvested from Haripur exhibited 1.72-fold higher SSC: TA ratio

than from Lahore. However, SSC: TA ratio did not show significant difference among

cultivars (Table 4.2).


54

Table 4.1 Effect of harvest locations and cultivars on weight loss, SSC, TA and SSC: TA ratio of litchi fruit during shelf period

Locations

Cultivars

Weight loss (%) SSC (oBrix) TA (%) SSC: TA Shelf period (Days) Shelf period (Days) Shelf period (Days) Shelf period (Days) 1 3 5 1 3 5 1 3 5 1 3 5

Lahore ‘Gola’ 0.0 15.0b 20.9c 14.2f 14.4f 17.43e 0.30ab 0.17cd 0.10de 46.2d 88.9d 196.6c

‘Serai’ 0.0 17.2f 23.3a 17.4e 19.7bc 23.3a 0.33a 0.30ab 0.10de 50.0d 66.0d 183.8c

Haripur ‘Gola’ 0.0 9.2e 22.3ab 17.3e 18.3cde 17.0e 0.27ab 0.23bc 0.03e 67.6d 81.7d 482.6a

‘Serai’ 0.0 17.6d 22.1ab 17.6de 19.3bcd 20.1b 0.30ab 0.33a 0.10de 57.9d 56.7d 339.1b

LSD value (P≤0.05) Locations Cultivars Shelf period Locations × Cultivars × Shelf period weight loss 0.8194 0.8194 1.0036 2.0071 SSC 0.7405 0.7405 0.9069 1.8139 TA 0.0281 0.0281 0.0344 0.0688 SSC:TA 38.687 38.687 47.382 94.763

The means sharing the same letter are non-significant at P≤0.05, Days = number of days after harvest, SSC = soluble solid concentrations, TA = titratable acidity


55

Table 4.2 Mean effect of harvest locations and cultivars on physiological weight loss, browning index and biochemical quality

attributes and enzymatic activities in peel tissues of litchi fruit

Parameter

Harvest locations LSD Value P ≤ 0.05

Cultivars LSD Value P ≤ 0.05 Lahore Haripur Serai Gola

Weight loss (%) 12.70a 11.30b 0.8194 12.86a 11.23b 0.8194 Pericarp browning 3.99a 3.42b 0.2429 3.76a 3.66a NS SSC (Brix°) 17.74a 18.27a NS 19.58a 16.43b 0.7405 TA (%) 0.22a 0.21a NS 0.24a 0.18b 0.0281 SSC:TA 105.25b 180.86a 38.687 125.51a 160.60a NS Ascorbic acid (mg 100 g-1) 48.89a 30.83b 3.5253 41.39a 38.33a NS TP (mg GAE 100 g-1) 187.35b 234.18a 3.5224 202.91b 218.62a 3.5224 Total antioxidants (%) 45.27b 58.00a 1.8947 50.21b 53.17a 1.8947 Anthocyanin 0.35a 0.334a NS 0.34a 0.35a NS CAT peel (U mg-1 Protein) 25.87a 27.05a NS 22.32b 30.60a 3.2340 POD peel (U mg-1 Protein) 36.74a 30.62b 4.0498 35.81a 31.55b 4.0498 SOD peel (U mg-1 Protein) 40.86b 52.16a 3.6937 39.85b 53.17a 3.6937 PPO peel (U mg-1Protein) 26.26a 21.88b 1.3813 25.22a 22.91b 1.3813

Means followed by different letters for a given parameter are significantly different at P ≤ 0.05 (LSD test). NS = non-significant (P ≤ 0.05). SSC = soluble solid concentration, TA = titratable acidity, TP = total phenolics, CAT = catalase, POD = peroxidase, SOD = superoxide dismutase, PPO = polyphenol oxidase


56


Significant decrease in TP of peel tissues was observed in both cultivars and locations

as number of days at shelf period progressed. On day-1 of shelf period highest amount of TP

were recorded in both cultivars and locations and minimum was recorded at day-5 of shelf

period (Fig. 4.2A and C). Overall fruit of litchi cv. ‘Gola’ exhibited significantly about 1.1-

fold higher TP than ‘Serai’ in peel tissues of litchi fruit (Table 4.2). Fruits harvested from

Haripur exhibited 1.25-fold higher TP than harvested Lahore (Table 4.2). Similarly total

antioxidants in peel tissues also decreased significantly in both locations and cultivars as

number of days passed at ambient conditions. In both locations highest total antioxidants

were recorded on day-1 of shelf period and lowest were recorded on day-5 of shelf period

(Fig. 4.2B and D). There was significance difference in concentration of total antioxidants

with respect to locations as fruit harvested from Haripur showed almost 1.3-fold higher total

antioxidants than fruit harvested from Lahore (Table 4.2). Fruit of ‘Gola’ cultivar exhibited

significantly higher (1.05-fold) total antioxidants concentration, as compared to ‘Serai’ fruit

(Table 2).


57

Fig. 4.1 Effect of harvest locations and cultivars on pericarp browning (A, D), anthocyanin contents (B, E) and activity of polyphenol oxidase enzyme (C, F) in peel tissues of litchi fruit during shelf life at ambient conditions. Vertical bars represents ± SE of means.

Similar to peel tissues TP and total antioxidants were also decreased in pulp tissues of

litchi fruit. On day-1 of shelf period highest amount of TP were recorded in both cultivars

and locations and minimum was recorded at day-5 of shelf period. Fruits harvested from

Haripur exhibited 1.20-fold higher TP than Lahore in pulp tissues of litchi fruit (Fig. 4.3B

and E). Similarly total antioxidants in pulp tissues were also decreased significantly in both

locations and cultivars as number of days passed at ambient conditions. In both locations

highest total antioxidants were recorded on day-1 of shelf period and lowest were recorded


58

on day-5 of shelf period. There was significance difference in concentration of total

antioxidants with respect to locations as fruit harvested from Haripur showed almost 1.22-

fold higher total antioxidants than fruit harvested from Lahore. Overall fruit of ‘Gola’

cultivar exhibited significantly higher (1.25-fold) total antioxidants concentration, as

compared to ‘Serai’ fruit (Fig. 4.3C and F). Ascorbic acid in pulp tissues of litchi fruit were

decreased with increase in number of days at shelf period (Fig. 4.3A and D). Maximum

ascorbic acid was exhibited by litchi cv. ‘Serai’ (63.3 mg 100 g-1) harvested from Lahore on

day-1 of shelf period (Fig. 4.3A), whereas lowest ascorbic acid (25 mg 100 g-1) was recorded

on day-5 of shelf period in fruit harvested from Haripur (Fig. 4.3D). Fruit harvested from

Lahore showed 1.58-fold higher ascorbic acid than from Haripur (Table 4.2).

Activities of CAT, SOD and POD enzymes

Varying behaviour in the activity of SOD enzyme in peel tissues of litchi was noted

as SOD enzyme activity was increased till day-3 of shelf period, but later on decreased

significantly on day-5 of shelf period (Fig. 4.4A and D). Significant difference among

cultivars was observed as SOD enzyme activity was cumulatively 1.33-fold higher in fruit of

litchi cv. ‘Gola’ than ‘Serai’ (Table 4.2). Decreasing trend in CAT enzyme activity was

observed in peel tissues of litchi fruit as days at shelf period progressed (Fig. 4.4B and E).

Effect of location on CAT enzyme activity did not show any significant difference, whereas,

difference among cultivars was significant (Table 4.2). Overall fruit of litchi cultivar ‘Gola’

exhibited 1.37-fold higher CAT enzyme activity than ‘Serai’, irrespective of location of

harvest (Table 4.2). POD enzyme activity was increased significantly from day-1 to day-5 of

shelf period in peel tissues of litchi fruit (Fig. 4.4C and F). Overall fruit of ‘Gola’ exhibited

1.13-fold lower POD enzyme activity, as compared to ‘Serai’ (Table 4.2) and fruit harvested

from Haripur showed 1.2-fold less POD enzyme activity, as compared to fruit harvested from

Lahore (Table 4.2).

Like peel tissues similar behaviour in the activities of SOD, CAT and POD enzymes

was also observed in pulp tissues of litchi fruit. Activity of SOD was increased till day-3, but

later on it was decreased and minimum activity was recorded on day-5. Significant difference

among cultivars was observed as SOD enzyme activity was cumulatively 1.11-fold higher in


59

fruit of litchi cv. ‘Gola’ than ‘Serai’ (Fig. 4.5A and D). Decreasing trend in CAT enzyme

activity was observed as days at shelf period progressed (Fig. 4.5B and E). Effect of location

on CAT enzyme activity did not show any significant difference, whereas, difference among

cultivars was significant. Overall fruit of litchi cultivar ‘Gola’ exhibited 1.18-fold higher

CAT enzyme activity than ‘Serai’, irrespective of location of harvest (Fig. 4.5B and E). POD

enzyme activity was increased significantly from day-1 to day-5 of shelf period. Overall fruit

of ‘Gola’ exhibited 1.12-fold lower POD enzyme activity, as compared to ‘Serai’ and fruit

harvested from Haripur showed 1.73-fold less POD enzyme activity, as compared to fruit

harvested from Lahore (Fig. 4.5C and F).

Fig. 4.2 Effect of harvest locations and cultivars on total phenolics (A, C) and total antioxidants (B, D) in peel tissues of litchi fruit during shelf life at ambient conditions. Vertical bars represents ± SE

of means.


60

Fig. 4.3 Effect of harvest locations and cultivars on ascorbic acid (A, D), total phenolics (B, E) and total antioxidants (C, F) in pulp tissues of litchi fruit during shelf life at ambient conditions. Vertical

bars represents ± SE of means.


61

Fig. 4.4 Effect of harvest locations and cultivars on activities of superoxide dismutase (A, D), catalase (B, E) and peroxidase (C, F) enzymes in peel tissues of litchi fruit during shelf life at ambient conditions. Vertical bars represents ± SE of means.


62

Fig. 4.5 Effect of harvest locations and cultivars on activities of superoxide dismutase (A, D), catalase (B, E) and peroxidase (C, F) enzymes in peel tissues of litchi fruit during shelf life at ambient conditions. Vertical bars represents ± SE of means.


63

4.5 DISCUSSION

As expected fruit weight loss was increased significantly with increase in shelf

period. The increase in weight loss was probably due to moisture loss through transpiration.

Tendency of weight loss with increase in time period was also confirmed by Mitra and Kar

(2001) in litchi cv. ‘Bombai’. SSC is an estimate of sugar contents and determines eating

quality of fruit. SSC of the litchi fruit was increased irrespective to locations and cultivars

with increase in shelf period, which could be due to breakdown of starch into sugars (Akhtar

et al., 2010). Subsequent, moisture loss could have concentrated the soluble solids of the fruit

and resulted in higher SSC as storage period progressed (Tanada-Palmu and Grosso, 2005).

Results are also in agreement with Aklimuzzaman et al. (2011) who observed increase in

SSC with increase in storage duration in litchi cv. ‘Bedana’. Previously, similar results were

observed when SSC of different cultivars of peach fruit harvested from different locations

was increased (Ullah et al., 2013). TA showed continuous decline with progress of shelf

period in fruit harvested from both locations, due to which SSC: TA ratio was increased with

increase in shelf period. Decrease in TA might be ascribed to oxidation of organic acids

during respiration or due the metabolic changes in fruit (Gimnez et al., 2003; Shiri et al.,

2011).

Pericarp browning is one of the major postharvest problems in litchi which reduces its

commercial value. In the present study, linear increase in pericarp browning index was

observed in litchi cvs. ‘Serai’ and ‘Gola’ from day-1 to day-5 of shelf period at ambient

conditions. Whereas, anthocyanin contents were decreased regularly at ambient conditions

with increase in shelf period. This increase in pericarp browning and decrease in anthocyanin

contents might be ascribed to increase in the activities of POD and PPO enzymes. After

harvest during shelf period litchi fruit undergo stress with loss of moisture, breakdown of cell

membrane and increased activity of PPO enzyme. When PPO enzyme comes in contact with

anthocyanin in presence of oxygen, the anthocyanin are irreversibly complexed into melanin

by-products causing pericarp browning (Lin et al., 1988). Similarly, in the present study

degradation of anthocyanin contents might be ascribed to higher activities of POD and PPO

enzymes and these results were also in line with data of Zauberman et al. (1991). However,

the difference in anthocyanin contents in both cultivars could be due to difference in genetic


64

make-up of cultivars which control anthocyanin level as previously reported in lettuce

(Mathew et al., 2005). Activity of PPO enzyme was increased from day-1 to day-5 of shelf

period. As at ambient conditions humidity was low due to which pH might increase and PPO

enzyme became active. These results are in accordance with Jiang (2000), who observed

maximum activity of PPO enzyme at 6.8 pH and zero activity below 4.0 pH in litchi cv.

‘Huaizhi’ fruit. Significant reduction in ascorbic acid of both litchi cvs. (‘Gola’ and ‘Serai’)

was observed. Ascorbic acid vary in different cultivars due to changing micro-climatic

conditions such as warm days and cold nights, as temperature also affects the ascorbic acid

accumulation (Lee and Kader, 2000; Mozafar, 1994; Shewfelt, 1990). Decrease in ascorbic

acid with increase shelf period could be due the oxidation of organic acids (Gimnez et al.,

2003). TP decreased continuously in peel and pulp tissues of litchi fruit from day-1 to day-5

of shelf period. This reduction may be attributed to the high rate of oxidation of TP by PPO

enzyme with progress in shelf period. These results confirm the findings of Altunkaya and

Gokmen (2008) where decline in TP of lettuce was observed due to oxidation by PPO

enzyme. Level of total antioxidants in peel and pulp tissues was decreased continuously from

day-1 to day-5 of shelf period but cv. ‘Gola’ exhibited higher antioxidants level compared to

cv. ‘Serai’ fruit. Many factors may alter antioxidant capacity of fruit including geographical

locations and cultivars. Difference in distribution pattern of metabolic tissues could be the

reason of difference in antioxidant capacity among cultivars (Shewfelt, 1990). The reduction

in total antioxidants at ambient conditions with increase in shelf period might be due to

oxygen stimulated oxidation of ascorbic acid and phenolic compounds (Stewart et al., 1999).

Activities of CAT and SOD enzymes were also decreased in peel and pulp tissues as

number of days at shelf progressed. The decrease in CAT enzyme activity may be due to the

prolonged oxidative stress that fruit had faced at ambient conditions during shelf period.

SOD and CAT enzyme are directly related to the senescence of the fruit, as fruit deteriorated

rapidly and peel turned brown, activities of SOD and CAT enzyme were also decreased till

day-5 of shelf period, resulting in the pericarp browning of the fruit and lowering the quality

of aril (Sun et al., 2010). Activities of POD enzyme in pulp and peel tissues were increased

from day-1 to day-5 of shelf period at ambient conditions. Moisture loss causes increase in

pH and increasing pH caused activation of POD enzyme. These results are also supported by


65

data from Mizobutsi et al. (2010) who observed that POD enzyme activity could be inhibited

more effectively at acidic pH rather than at alkaline pH.

4.6 Conclusion

Cultivars and harvest locations significantly influenced pericarp browning and

physico-chemical attributes along with the activities of antioxidative enzymes in litchi fruit.

Cultivar ‘Gola’ exhibited superior fruit quality characteristics than ‘Serai’, while Haripur

location produced fruit with better quality than Lahore.

Chapter‐5 Role of oxalic acid on pericarp browning and quality of litchi

66

Chapter 5

Exogenous Application of Oxalic Acid Delay Pericarp Browning and Maintain Quality of Litchi cv. ‘Gola’ Fruit

5.1 Abstract

Pericarp browning is a major limiting factor affecting postharvest life and quality of

litchi fruit. Therefore, the present study was conducted to investigate the influence of oxalic

acid on pericarp browning and changes in the biochemical quality attributes in litchi cv.

‘Gola’ fruit. Fruit harvested at physiological maturity were dipped in different concentrations

of oxalic acid (0, 1, 2, 3 or 4 mM) with tween 20 (0.01%) as a surfactant and stored for 28

days at 5 ± 1oC with 90-95% RH. Physical (browning index and weight loss), biochemical

[soluble solid concentrations (SSC), titratable acidity (TA), SSC: TA ratio, ascorbic acid,

anthocyanin contents, total phenolics (TP) and total antioxidants] fruit quality and activities

of enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and polyphenol

oxidase (PPO)] were determined at 7 days interval during cold storage. Postharvest

application of 2 mM oxalic acid reduced fruit weight loss and delayed pericarp browning by

maintaining higher anthocyanin contents (0.32 ∆Ag-1 FW), as compared to control. SSC and

ascorbic acid were higher in fruit treated with 2 mM oxalic acid, in contrast to untreated fruit.

Activities of PPO and POD enzymes in litchi peel tissues were reduced in fruit treated with 2

mM oxalic acid during 28 days of cold storage. Activities of antioxidative enzymes (SOD

and CAT) and level of TP and total antioxidants in litchi peel as well as pulp tissues were

significantly higher in 2 mM oxalic acid-treated fruit. In conclusion, postharvest application

of 2 mM oxalic acid significantly delayed pericarp browning and maintained better quality of

‘Gola’ litchi fruit during cold storage.

5.2 Introduction

Litchi is an important commercial fruit crop and is believed to be originated from

northern Vietnam and southern China (Menzel, 2001). It has high market value due to

deliciously flavoured translucent juicy aril which contains sufficient amount of vitamins and

mineral (Wall, 2006). Postharvest pericarp browning is the main issue in litchi fruit which

Chapter‐5 Role of oxalic acid on pericarp browning and quality of litchi fruit

67

involves rapid loss of red colour after harvest (Kumar et al., 2013), thus affecting the fruit

quality and hindering its market potential (Rajwana et al., 2010).

The red colour of litchi peel starts getting darker, when oxidative enzymes in

cytoplasm react with phenolic substrates present in vacuole through assistance of plastid

membrane structure. Browning takes place when PPO and phenolic substrates are provided

with optimum oxygen, temperature and pH (De-Reuck et al., 2009). During storage,

consistent water loss brings biochemical and physiological changes such as reduction of

membrane integrity, degradation of anthocyanins, increase in the activities of POD and PPO

enzymes, and finally enhances superoxidation reaction causing pericarp browning (Jiang,

2000; Lima et al., 2010).

Previously SO2 was used commercially to control pericarp browning in litchi, but

sulphur residues left in fruit caused harmful effects on consumer’s health (Sivakumar et al.,

2005; Kudachikar et al., 2007). Therefore, other substitutes such as citric acid with cassava

starch, hydrochloric acid with PVC film, chitosan in combination with ascorbic acid and N-

Acetyl Cysteine have also been reported to delay pericarp browning in litchi (Liu et al., 2006;

Silva et al., 2012; Sun et al., 2010). Similarly, various anti-browning agents like calcium

chloride, Iso-ascorbic acid, 4-hexylresorcinol and citric acid have also been found effective

to control browning in apple, pear, loquat and mango (Monsalve-Gonzalez et al., 1995; Buta

and Abbott, 2000; Ding et al., 2002; Guerrero-Beltran et al., 2005). Earlier, postharvest

application of oxalic acid was reported to control browning in apple and banana slices by

decreasing PPO activity (Yoruk et al., 2002). Similarly, oxalic acid-treated litchi cv.

‘Huaizhi’ fruit exhibited delayed pericarp browning when kept at ambient conditions (Zheng

and Tian, 2006).

It appears that application of oxalic acid can effectively control the pericarp

browning, however, to the best of my knowledge its effect on pericarp browning under

prolonged cold storage has not yet been investigated. Therefore, present study was conducted

to further explore the anti-browning characteristics of oxalic acid and standardize its

optimum concentration to control pericarp browning in commercial litchi cultivar ‘Gola’

under extended cold storage conditions.


68


Treatments

Fruit of litchi cv. ‘Gola’ were harvested from Fruit Farm Nursery, Haripur. Fruit of

uniform, size, shape, free from any physical damage, packed in corrugated fiberboard boxes

were transported to Postharvest Research and Training Centre (PRTC), Institute of

Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan using reefer van

(Section 3.1.1; page 34). Fruit were dipped for 5 min in an aqueous solution containing

different concentrations of oxalic acid (0, 1, 2, 3 or 4 mM) with ‘Tween 20’ (0.01%) as a

surfactant and then stored at 5 ± 1oC with 90 ± 5% RH for 28 days. The fruit were removed

at 7 days interval from cold storage to determine fruit weight loss (%), pericarp browning

index; whereas, SSC, TA, SSC: TA ratio, ascorbic acid were determined from juice samples

obtained from pulp tissues. Moreover, TP, total antioxidants, activities of POD, CAT and

SOD enzymes were also determined at 7 days interval during cold storage in both peel as

well as pulp tissues of litchi fruit. However, activity of PPO and anthocyanin contents was

determined only from peel tissues. The study was conducted under completely randomized

design with two factors factorial arrangement (i.e. oxalic acid concentration and storage

period) having 25 fruit as an experimental unit replicated three times.

Pericarp browning

Pericarp browning was assessed by using the method outlined in detail in section


Fruit weight loss








69






















Statistical Analysis


Statistix 10 for Windows software with two-factor factorial arrangements including oxalic

acid treatments and cold storage period. The effects of treatments were determined from the

least significant differences test (Fisher’s LSD) at P ≤ 0.05, where the F test was significant

(Steel et al., 1997). Pearson’s correlations were also performed to estimate relationship


70

between fruit browning index, anthocyanin contents and various antioxidative enzyme

activities using Statistix 10 (Analytical Software, Tallahasee, FL 32317, USA).

5.4 Results

Fruit weight loss, pericarp browning, anthocyanin contents and PPO activity

Fruit weight loss was significantly increased with the progression of storage period.

Fruit weight loss during cold storage was low in oxalic acid-treated fruit, as compared to

control (Fig. 5.1A). Fruit treated with 2 mM oxalic acid exhibited 2.03-fold lower fruit

weight loss than untreated fruit after 28 days of cold storage (Fig. 5.1A). As expected,

pericarp browning index increased during cold storage in both oxalic acid-treated and control

fruit. Control fruit showed 1.36-fold higher browning index, as compared to 2 mM oxalic

acid-treated fruit after 28 days of cold storage. However, all oxalic acid treatments retarded

the pericarp browning in litchi cv. ‘Gola’ during 28 days of cold storage (Fig. 5.1B). Among

treatments, 2 mM oxalic acid-treated fruit exhibited 1.06-fold less pericarp browning than 3

mM oxalic acid-treated fruit. Anthocyanin contents in peel tissues of litchi fruit deteriorated

rapidly as they were negatively correlated (r = -0.84) with pericarp browning (Table 5.1).

Decrease in anthocyanin contents of litchi peel tissues was significantly higher (3.37-fold) in

control fruit than 2 mM oxalic acid-treated fruit after 28 days of cold storage. Among

treatments, fruit treated with 2 mM oxalic acid exhibited 2.30-fold higher anthocyanin

contents. Activities of PPO enzymes in peel tissue increased significantly during cold storage

(Fig. 5.1D). In control fruit activity of PPO enzyme was significantly (1.47-fold) higher, as

compared to oxalic acid-treated fruit during 28 days of cold storage. However, among the

oxalic acid treatments, maximum PPO enzyme activity was observed in fruit treated with 1

mM oxalic acid which was 1.19-fold higher than fruit treated with 2 mM oxalic acid after 28

days of cold storage. Activities of PPO enzyme in peel tissues showed significant positive

correlation (r = 0.95) with pericarp browning index during cold storage (Table 5.1).


71

Fig. 5.1 Effect of different concentrations of postharvest application of oxalic acid on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase enzyme (D) in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


72

Table 5.1. Relationship of pericarp browning index with activities of PPO, POD, SOD, CAT enzymes, TP and total antioxidants in oxalic acid-treated litchi peel and pulp tissues.

Parameter Peel Pulp

PBI vs anthocyanin contents -0.8407** ---

PBI vs PPO 0.9489** ---

PBI vs POD 0.9207** 0.9126**

PBI vs SOD -0.9249** -0.8950**

PBI vs CAT -0.9181** -0.9052**

PBI vs TP -0.9656** -0.9545**

PBI vs total antioxidants -0.8119* -0.8955**

PBI = Pericarp browning index, PPO = polyphenol oxidase, POD = peroxidase, TP = total phenolics, SOD = superoxide dismutase, CAT = catalase, ** = significant at P ≤ 0.01.

SSC, TA and SSC: TA

SSC was significantly increased as the storage period progressed, irrespective to

treatments. After 28 days of cold storage, control fruit showed about 1.06-fold lower SSC, as

compared to 2 mM oxalic acid-treated fruit. Among oxalic acid concentrations, 2 mM oxalic

acid-treated fruit exhibited highest (1.04-fold) SSC than 4 mM oxalic acid-treated fruit after

28 days of cold storage (Fig. 5.2A). Continuous decline in TA was observed during entire

cold storage period. Control fruit showed quick decline in TA which was1.35-fold more, as

compared to oxalic acid-treated fruit. Among oxalic acid-treated fruit, lowest TA (1.10-fold)

was observed in fruit treated with 1 mM oxalic acid than 2 mM oxalic acid after 28 days of

cold storage (Fig. 5.2B). With the advancement of cold storage period, linear increase in

SSC: TA ratio was observed, irrespective to treatments. Control fruit showed about 1.46-fold

higher SSC: TA ratio, as compared to 4 mM oxalic acid treatment after 28 days of cold

storage. Fruit dipped in 1 mM oxalic acid solution resulted in about 1.25-fold higher SSC:

TA ratio than 4 mM oxalic acid-treated fruit after 28 days of cold storage (Fig. 5.2C).


73

Fig. 5.2 Effect of different concentrations of postharvest application of oxalic acid on SSC (A), TA (B) and SSC: TA (C) ratio in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


74

TP, total antioxidants and ascorbic acid

Irrespective to treatments, litchi cv. ‘Gola’ fruit showed linear decreasing trend for TP

and total antioxidants in the peel tissues during entire cold storage period (Fig. 5.3). Fruit

treated with 2 mM oxalic acid exhibited significantly about 1.57-fold higher TP, as compared

to control fruit after 28 days of cold storage (Fig. 5.3A). Total antioxidants were decreased

significantly with increase in storage period, irrespective to treatment. However, 2 mM

oxalic acid-treated fruit exhibited about 1.38-fold higher level of total antioxidants than

control fruit (Fig. 5.3B). Among oxalic acid concentrations, fruit treated with 2 mM oxalic

acid showed significantly higher (1.11-fold) level of total antioxidants than 1 mM oxalic

acid-treated fruit after 28 days of cold storage (Fig. 5.3B). Pericarp browning index exhibited

negative correlation with TP and total antioxidants in both peel (r = -0.96; r = -0.81) and pulp

tissues (r = -0.95; r = -0.89), respectively (Table 5.1).

Similar to peel tissues, gradual decline in the levels of TP, total antioxidants and

ascorbic acid were also observed in pulp tissues of litchi fruit with the advancement of cold

storage period (Fig. 5.4A and B). Oxalic acid-treated fruit (2 mM) exhibited 2.10- and 1.70-

fold higher TP and total antioxidants in pulp tissues, respectively than control fruit during 28

days of cold storage. However, after 28 days of cold storage, fruit treated with 2 mM oxalic

acid showed significantly about 1.25- and 1.17-folds higher TP and total antioxidants in pulp

tissues, respectively than 4 mM oxalic acid-treated fruit (Fig. 5.4A and B). Ascorbic acid in

pulp tissues also showed continuous decline during entire storage period. Decline in ascorbic

acid in pulp tissues was 1.48-fold higher in untreated fruit after 28 days of cold storage than 2

mM oxalic acid-treated fruit. Among different oxalic acid treatments, fruit treated with 2 mM

oxalic acid exhibited 1.15-fold higher ascorbic acid in pulp tissues after 28 days of cold

storage than 1 mM oxalic acid-treated fruit (Fig. 5.4C).


75

Fig. 5.3 Effect of different concentrations of postharvest application of oxalic acid on total phenolics (A) and total antioxidants (B) in litchi peel tissues during cold storage. Vertical bars represents ± SE of means.


76

Fig. 5.4 Effect of different concentrations of postharvest application of oxalic acid total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.

Chapter‐5 Role of oxalic acid on pericarp browning and quality of litchi

77

Fig. 5.5 Effect of different concentrations of postharvest application of oxalic acid on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues litchi of litchi fruit during cold storage. Vertical bars represents ± SE of means.


78

Fig. 5.6 Effect of different concentrations of postharvest application of oxalic acid on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


79

SOD, CAT and POD activities

A linear decline in the activities of SOD and CAT enzymes in peel tissues of litchi

fruit was observed during entire storage period; however, oxalic acid-treated fruit maintained

higher activities of these enzymes (Fig. 5.4A and B). Activities of SOD and CAT enzymes

were significantly about 1.80- and 1.86-folds lower in control fruit, respectively, as compared

to 2 mM oxalic acid-treated fruit after 28 days of cold storage. Fruit treated with 2 mM oxalic

acid exhibited 1.12-fold higher activities of SOD enzymes, as compared to 1 mM oxalic acid-

treated fruit and 1.42-fold higher activity of CAT enzyme than 4 mM oxalic acid-treated fruit

after 28 days of cold storage. Pericarp browning index was negatively correlated with the

activities of SOD (r = -0.92) and CAT (r = -0.91) enzymes in peel tissues of litchi fruit

during cold storage (Table 5.1). On the other hand, increasing trend in POD activity was

observed in peel tissues during cold storage. Control fruit showed significantly higher (1.78-

fold) activity of POD enzyme than oxalic acid-treated fruit after 28 days of cold storage.

Application of oxalic acid (3 mM) showed 1.35-fold higher activities of POD enzymes in

litchi peel tissues than 2 mM oxalic acid-treated fruit after 28 days of cold storage (Fig.

5.5C). Oxalic acid-treated fruit exhibited positive correlation (r = 0.92) between pericarp

browning index and activities of POD in peel tissues (Table 5.1).

Similarly, substantial decline in the activities of SOD and CAT enzymes were

observed in pulp tissues of litchi fruit with advancement in cold storage period. Control fruit

exhibited significantly about 2.21- and 1.49-fold lower activities of SOD and CAT enzymes

than oxalic acid-treated fruit (Fig. 5.6A and B). On the other hand, after 28 days of storage,

fruit treated with 2 mM oxalic acid showed significantly higher activities of SOD (1.38-fold)

and CAT (1.31-fold) enzymes than 3 mM and 1 mM oxalic acid-treated fruit (Fig. 5.6A and

B). Increasing trend was observed in the activity of POD enzyme in pulp tissues of litchi fruit

(Fig. 5.6C). Control fruit exhibited 1.62-fold higher activity of POD after 28 days of cold

storage, as compared to 2 mM oxalic acid-treated fruit. However, after 28 days of cold

storage 2 mM oxalic acid-treated fruit exhibited 1.16-fold lower activity of POD enzyme

than other oxalic acid treatments. Activities of SOD and CAT enzymes exhibited negative


80

correlations (r = 0.90; 0.91) and POD enzyme showed positive correlation (r = 0.91) with

pericarp browning index in pulp tissues of oxalic acid-treated fruit (Table 5.1).

5.5 Discussion

As expected fruit weight loss was increased significantly with increase in cold storage

period. The increase in weight loss was probably due to moisture loss through transpiration,

respiration and various metabolic activities taking place in fruit with progression of cold

storage period (Narayana et al., 1996). Tendency of weight loss with increase in storage

period was also confirmed by Mitra and Kar (2001) in ‘Bombai’ litchi. However, reduced

fruit weight loss in oxalic acid-treated fruit might be due to maintenance of membrane

integrity and low respiration rate which consequently slowed down metabolic activities and

ultimately helped in moisture retention. Similarly, oxalic acid-treated pomegranate fruit

showed less fruit weight loss when stored at 2°C for 84 days (Sayyari et al., 2010). Gradual

increase in pericarp browning was observed in ‘Gola’ litchi fruit with increase in weight loss,

as reported by Joas et al. (2005) in ‘Kwai Mi’ litchi. However, oxalic acid-treated fruit

exhibited less pericarp browning and high anthocyanin contents than control fruit during cold

storage period. Similarly, exogenous application of oxalic acid resulted in lower pericarp

browning index and higher anthocyanin contents in ‘Huaizhi’ litchi when kept at ambient

conditions (Zheng and Tian, 2006). In litchi, higher activities of PPO and POD enzymes are

considered to cause pericarp browning (Jiang et al., 2006; Underhill and Critchley, 1995).

Oxalic acid application significantly reduced the activities of PPO and POD enzymes

in peel tissues by maintaining low acidic pH as activity of PPO was inhibited below pH 4.0

in litchi fruit (Jiang, 2000). Oxalic acid-treated fruit maintained high anthocyanin contents

throughout the cold storage period. Oxalic acid being a natural antioxidant preserved

anthocyanin contents from degradation and conversion into brown colour by-products

(Zheng and Tian, 2006). Oxalic acid-treated fruit exhibited negative correlation between

anthocyanin contents in peel tissues and pericarp browning. Previously, negative correlation

between pericarp browning and anthocyanin contents has also been reported in longan fruit

(Lin et al., 2005). Degradation of anthocyanin contents might be ascribed to higher activities

of POD and PPO enzymes (Zauberman et al., 1991), as both enzymes showed higher


81

activities with increase in cold storage period. However, oxalic acid-treated fruit exhibited

lower activities of PPO and POD enzymes in peel tissues than untreated fruit. Oxalic acid

application might have reduced the pH; therefore, PPO activity was low in oxalic acid-treated

fruit as activity of PPO enzyme is triggered by moisture loss and change in pH from acidic to

basic. Similarly, the peak activity of PPO enzyme was observed at pH 6.8, while, at pH 4.0

its activity was not detectable in ‘Huaizhi’ litchi (Jiang, 2000).

SSC was increased in pulp tissues of litchi fruit with progression of cold storage

period, irrespective to treatments. However, oxalic acid application resulted in less increase

in SSC than control fruit. Oxalic acid might have increased membrane stability (Zhang et al.,

2001b), and developed resistance to moisture loss due to which increase in SSC of the treated

fruit was lower than control. Previously, similar results were also reported by Rub et al.

(2010) in sweet orange, where moisture loss resulted in higher SSC. Hydrolysis of starch into

sugars could be the reason of increase in SSC (Mahajan and Goswami, 2004). Increase in

SSC with the progress of storage period was also reported by Aklimuzzaman et al. (2011)

and Rajak et al. (2014) in ‘Bedana’ and ‘Shahi’ cvs. of litchi, respectively. Similarly,

increasing trend in SSC rate was also observed in grapes berries by Tanada-Palmu and

Grosso (2005). TA showed continuous decline with extended storage period in pulp tissues,

due to which SSC: TA ratio was increased with progression of storage period. However,

oxalic acid-treated fruit maintained high TA than untreated fruit, as oxalic acid might have

reduced respiration rate and other metabolic activities responsible for converting organic

acids into sugars. Previously, similar trend of TA was observed in oxalic acid-treated mango

fruit, where oxalic acid application maintained higher TA in contrast to untreated fruit

(Zheng et al., 2007a).

TP in litchi peel tissues were decreased continuously during 28 days of cold storage

but oxalic acid application reduced the decline in TP, as compared to untreated fruit. TP were

negatively correlated with pericarp browning index in peel tissues of litchi fruit and similar

correlation was also reported by Lin et al. (2005) in longan fruit. This reduction in TP might

be attributed to the high rate of oxidation of phenolic compounds by PPO enzyme with

progression of cold storage period. These results are similar to the findings of Altunkaya and


82

Gokmen (2008) where decline in TP of lettuce was observed due to oxidation of PPO. Oxalic

acid-treated fruit maintained higher TP in peel tissues than untreated fruit by reducing the

rate of oxidation of phenolic compounds by activities of PPO and POD enzymes. Total

antioxidants in litchi peel tissues were decreased continuously with increase in cold storage

period. However, oxalic acid-treated fruit maintained higher level of total antioxidants than

untreated fruit. Total antioxidants of litchi peel tissues showed significant negative

correlation with pericarp browning index. The reduction in total antioxidants in litchi peel

tissues with increase in storage period might be due to oxygen stimulated oxidation of

ascorbic acid or phenolic compounds and similar reduction in antioxidants was also observed

in strawberry fruit (Stewart et al., 1999). Similarly, in litchi pulp tissues level of TP and total

antioxidants was decreased with advancement in storage period. TP and total antioxidants

help in improving the antioxidative capacity of the fruit (Duthie and Crozier, 2000), that’s

why due to reduction of TP in pulp tissue, antioxidative capacity of litchi pulp was also

decreased. Previously, postharvest oxalic acid application has been reported to increase

phenolics and antioxidants potential of pomegranate stored at 2°C for 84 days (Sayyari et al.,

2010). Significant reduction in the ascorbic acid of litchi pulp tissues was noticed with

progression of cold storage period. However, decline in ascorbic acid was more pronounced

in untreated fruit in comparison with oxalic acid-treated fruit. Decrease in ascorbic acid

might be ascribed to respiration and other metabolic changes taking place in fruit resulting in

conversion of organic acids into sugars (Gimnez et al., 2003; Shiri et al., 2011). Additionally,

oxalic acid as a natural antioxidant suppressed the lipid peroxidation and ultimately

decreased the oxidation of ascorbic acid (Kayashima and Katayama, 2002).

SOD and CAT enzymes play important role under stress conditions, but their

activities were decreased in litchi peel and pulp tissues, as cold storage period progressed.

Activities of SOD and CAT enzymes were negatively correlated with pericarp browning and

similar correlation was also observed in longan fruit (Lin et al., 2005). However, oxalic acid-

treated fruit maintained higher activities of SOD and CAT enzymes than untreated fruit. Fruit

naturally contain SOD and CAT enzymes as a defense system against ROS (Niranjana et al.,

2009). These enzymes play major role in antioxidant defense system by maintaining the ROS

production below the threshold levels (Rao et al., 1996). Higher activities of CAT and SOD


83

enzymes in oxalic acid-treated fruit resulted in increased antioxidative capacity of fruit

against oxidative stress, thereby, delayed senescence and pericarp browning of litchi fruit

through inhibition of lipid peroxidation (Zheng and Tian, 2006).

Similar results were reported by Zheng et al. (2007b) when oxalic acid-treated peach

fruit kept at ambient condition maintained higher activities of SOD and CAT enzymes, as

compared to control fruit. On the other hand, activities of POD enzyme in peel and pulp

tissues of litchi were increased with the advancement of cold storage period. In litchi POD

enzyme is known to cause pericarp browning (Underhill and Critchley, 1995). However, our

results showed that oxalic acid-treated fruit maintained low activity of POD enzyme than

untreated fruit. These results were also supported by data from Zheng and Tian (2006) who

reported lower POD enzyme activity in oxalic acid-treated litchi fruit, as compared to

untreated fruit. Positive correlation between pericarp browning and activities of POD enzyme

in peel and pulp tissues of litchi was observed. Similar correlation of POD enzyme with

pericarp browning was also reported by Lin et al. (2005) in longan fruit.

5.6 Conclusion

Exogenous application of 2 mM oxalic acid delayed pericarp browning, maintained

better physico-chemical attributes along with the higher activities of antioxidative enzymes

(SOD and CAT) in litchi cv. ‘Gola’ fruit during cold storage.

Chapter‐6 Effect of ascorbic acid on pericarp browning and quality of litchi fruit

84

Chapter 6

Postharvest Application of Ascorbic Acid Delay Pericarp Browning and Enhance Antioxidative Capacity of Litchi cv. ‘Gola’ Fruit

6.1 Abstract

Pericarp browning is the major problem which affects the visual appearance and

quality of the litchi fruit. Therefore, present research was conducted to investigate the effects

of postharvest application of ascorbic acid on pericarp browning, changes in the activities of

antioxidative enzymes and biochemical quality attributes of litchi ‘Gola’ fruit. Fruit were

harvested at physiological maturity, dipped in different ascorbic acid concentrations (0, 15,

30, 45 or 60 mM) with 0.01% ‘Tween 20’ as a surfactant were stored at 5 ± 1°C with 90 ±

5% RH for 28 days. Physical (browning index and fruit weight loss), biochemical quality

characteristics [soluble solid concentrations (SSC), titratable acidity (TA), SSC: TA ratio,

ascorbic acid] were determined from pulp juice samples, whereas, anthocyanin contents were

determined in peel tissues. On the other hand, total phenolics (TP), total antioxidants,

activities of enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and

polyphenol oxidase (PPO)] were determined in peel and pulp tissues of litchi at 7 days

interval during cold storage. Postharvest application of ascorbic acid significantly reduced

fruit weight loss, pericarp browning by maintaining higher anthocyanin contents. SSC and

TA were higher in ascorbic acid-treated fruit, 15 and 30 mM, respectively, while ascorbic

acid and SSC: TA ratio was more in 45 mM ascorbic acid-treated fruit. Total phenolics and

total antioxidants contents were significantly higher in peel and pulp tissues of 45 mM

ascorbic acid-treated fruit. Activities of antioxidative enzymes (SOD and CAT) and level of

TP and total antioxidants in litchi peel and pulp tissues were significantly higher in 45 mM

ascorbic acid-treated fruit. Activities of PPO and POD enzymes in litchi peel tissues were

reduced in 45 mM ascorbic acid-treated fruit. Conclusively, postharvest application of 45

mM ascorbic significantly delayed pericarp browning and maintained better quality of ‘Gola’

litchi fruit during cold storage.


85

6.2 Introduction

Litchi is a tropical and subtropical fruit having a very high commercial value in the

international market (Jiang et al., 2004). Litchi fruit has a bright-red pericarp and a

translucent pulp of interesting nutritional value (Salomao et al., 2006). However, it has a very

short shelf life due to the rapid postharvest pericarp browning, as fruit turns brown within 72

h after harvest (Del-Aguila et al., 2009).

Pericarp browning in litchi fruit has been attributed to moisture loss through heat

stress, senescence, chilling injuries, pests and diseases. Skin browning is accompanied by

several changes that commonly results from the degradation of the anthocyanin pigments by

activation of different enzymes particularly polyphenol oxidase (PPO), peroxidase (POD)

and ascorbic acid oxidase (Mizobutsi et al., 2010) or by free radicals such as superoxide (O-

2), hydrogen peroxide (H2O2) and hydroxyl (-OH) radicals known as reactive oxygen species

(ROS) (Wang et al., 2007; Ye, 2005). ROS production may be due to failure of antioxidative

enzyme system to eliminate these free radicals (Kanazawa et al., 2000). Excessive production

of ROS can damage proteins, lipids and consequently leads to the loss of membrane integrity

and functionality (Yang et al., 2008).

Pericarp browning in litchi fruit can be delayed by using different anti-browning

agents. Previously, various chemical treatments including fumigation of sulfur dioxide

(Ducamp-Collin et al., 2008), exogenous application of different acid combined with edible

coatings such as hydrochloric acid (Jiang et al., 2004), ascorbic acid (Silva et al., 2010), citric

acid (Terdbaramee et al., 2003), oxalic acid (Saengnil et al., 2006) and edible coating of

chitosan combined with ascorbic acid have been reported to delay or prevent pericarp

browning in litchi fruit, however, with inconclusive and sporadic result (Zhang and Quantick,

1997; Sun et al., 2010).

Ascorbic acid as an antioxidant is known to prevent browning and delay senescence

in various fresh cut fruits and vegetables (Suttirak and Manurakchinakorn, 2010). Postharvest

application of ascorbic acid delayed pericarp browning of litchi fruit by maintaining

membrane integrity, thereby, inhibiting the leakage rate through suppressing the activities of


86

PPO and POD enzymes (Sun et al., 2010) or by the formation of ascorbyl which directly

scavenges ROS (Yamaguchi et al., 1999) and reduces the formation of o-quinones produced

by PPO (Robert et al., 2003). Fruit naturally have a defense mechanism against the oxidative

stress by the activation of antioxidant enzymes system such as catalase (CAT) and

superoxide dismutase (SOD) (Razzaq et al., 2013). Recently, the pulp tissues of fruit cv.

‘Feizixiao’ treated with ascorbic acid were showed to exhibit higher activities of SOD and

CAT during cold storage (Sun et al., 2010).

However, activities of antioxidative enzymes in peel and pulp tissues and their

relationship with pericarp browning index by the application of ascorbic acid is missing in

literature which further needs to be investigated. Therefore, it was hypothesized that

exogenous ascorbic acid application will reduce the pericarp browning by maintaining lower

activities of PPO and POD enzymes in peel and pulp tissues of litchi cv. ‘Gola’ fruit. Hence,

effect of postharvest application of ascorbic acid were investigated on fruit biochemical

properties and activities of the pericarp browning enzymes (PPO and POD), as well as other

antioxidative enzymes (SOD and CAT) in peel and pulp tissues of ‘Gola’ litchi fruit during

cold storage.


Treatments

Fruit of uniform size, shape and free from any physical damage were harvested from

30-35 year old uniform and healthy litchi trees of Fruit Farm Nursery, Haripur, KPK,

Pakistan. After harvest, fruit were packed in corrugated fiberboard boxes and were

transported to Postharvest Research and Training Centre (PRTC), Institute of Horticultural

Sciences, University of Agriculture, Faisalabad, Pakistan using reefer van as described in

detail in section 3.1.1 (page 34). Fruit of uniform size were dipped for 5 min in an aqueous

solution containing different concentrations of ascorbic acid (0, 15, 30, 45 or 60 mM) with

‘Tween 20’ (0.01%) as a surfactant and were stored at 5 ± 1oC with 90 ± 5% RH for 28 days.

The fruit were removed at 7 days interval from cold storage to determine fruit weight loss

(%), pericarp browning index; whereas, SSC, TA, SSC: TA ratio, ascorbic acid were


87

determined from juice samples obtained from pulp tissues. Moreover, TP, total antioxidants,

activities of POD, CAT and SOD enzymes were also determined at 7 days interval during

cold storage in both peel as well as pulp tissues of litchi fruit. However, activity of PPO

enzyme and anthocyanin contents was determined only from peel tissues. The study was

conducted under completely randomized design with two factors factorial arrangement (i.e.

ascorbic acid concentrations and storage period) having 25 fruit as an experimental unit

replicated three times.

Pericarp browning

Pericarp browning was evaluated by using the method outlined in detail in section


Fruit weight loss

















88







absorbance change of 0.01 unit min-1”. POD enzyme activity was determined by the guaiacol








Statistix 10 for Windows software with two-factor factorial arrangements including ascorbic

acid treatments and cold storage period. The effects of treatments were determined from the

least significant differences test (Fisher’s LSD) at P ≤ 0.05, where the F test was significant

(Steel et al., 1997). Pearson’s correlations were also performed to estimate relationship

between fruit browning index, anthocyanin contents and various antioxidative enzyme

activities using Statistix 10 (Analytical Software, Tallahasee, FL 32317, USA).

6.4 Results

Fruit weight loss, pericarp browning, anthocyanin and activity of PPO enzyme

Fruit weight loss was significantly increased with the progression of storage period.

All ascorbic acid treatments showed significantly less fruit weight loss during cold storage

compared to control fruit. Postharvest application of 45 mM ascorbic acid resulted in 2.29-

fold less fruit weight loss, as compared to control fruit. Fruit treated with 45 mM ascorbic


89

acid showed 1.50-fold less fruit weight loss than 30 mM ascorbic acid during 28 days of cold

storage (Fig. 6.1A).

As expected, pericarp browning index increased during storage in ascorbic acid-treated

and control fruit. However, ascorbic acid-treated fruit showed delay in pericarp browning by

exhibiting lower values of browning index during entire cold storage period. Control fruit

showed 1.48-fold higher browning index, as compared to 45 mM ascorbic acid-treated fruit

after 28 days of cold storage. After 28 days of cold storage, 45 mM ascorbic acid exhibited

1.20-fold less pericarp browning than 15 mM ascorbic acid-treated fruit (Fig. 6.1B).

Anthocyanin contents in peel tissues of litchi fruit deteriorated rapidly as they were

negatively correlated (r = -0.51) with pericarp browning (Table 6.1). Decline in anthocyanin

contents of litchi peel tissues was significantly higher (2.71-fold) in control fruit than in 45

mM ascorbic acid-treated fruit during 28 days of cold storage. However, after 28 days of

storage fruit treated with 45 mM ascorbic acid showed about 2.25-fold higher anthocyanin

contents, as compared to other treatments (Fig. 6.1C).

Activity of PPO in peel tissue increased significantly with progress in cold storage

period. However, control fruit showed significantly higher (1.27-fold) activity of PPO than

45 mM ascorbic acid-treated fruit exhibited after 28 days of cold storage. Among the

ascorbic acid treatments, fruit treated with 45 mM ascorbic acid exhibited significantly lower

activity of PPO (1.22-fold) than fruit treated with 30 mM ascorbic acid after 28 days of cold

storage (Fig. 6.1D). Activities of PPO in peel tissues showed significant positive correlation

(r = 0.76) with pericarp browning index during cold storage (Table 6.1).


90

Fig. 6.1 Effect of different concentrations of postharvest application of ascorbic acid on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase (D) in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


91

Table 6.1. Relationship of pericarp browning index with activities of PPO, POD, SOD, CAT enzymes, TP and total antioxidants in ascorbic acid-treated litchi peel and pulp tissues.

Parameter Peel Pulp

PBI vs anthocyanin Contents -0.511** ---

PBI vs PPO 0.762** ---

PBI vs POD 0.678** 0.772**

PBI vs SOD -0.810** -0.646**

PBI vs CAT -0.658** -0.793**

PBI vs TP -0.831** -0.815**

PBI vs total antioxidants -0.714** -0.6872**

PBI = pericarp browning index, PPO = polyphenol oxidase, POD = peroxidase, TP = total phenolics, SOD = superoxide dismutase, CAT = catalase, ** = significant at P ≤ 0.01.

SSC, TA and SSC: TA ratio

SSC of litchi fruit increased in all the treatments with the progression of cold storage

period. After 28 day of cold storage, 15 mM ascorbic acid-treated fruit showed about 1.07-

fold higher SSC, as compared to control fruit. Among ascorbic acid concentrations,

application of 15 mM ascorbic acid resulted in 1.05-fold more SSC than 30 mM ascorbic

acid-treated fruit (Fig. 6.2A). On the other hand, TA of litchi fruit juice showed continuous

decline during entire cold storage period. Control fruit showed quick decline in TA which

was about 1.37-fold higher, as compared to ascorbic acid-treated fruit. However, after 28

days of cold storage 30 mM ascorbic acid application resulted in 1.15-fold higher TA, as

compared to all other treatments (Fig. 6.2B). As storage period progressed, gradual increase

in SSC: TA ratio was observed, irrespective to treatments. Control fruit showed about 1.56-

fold higher SSC: TA ratio, as compared to 30 mM ascorbic acid treatment after 28 days

under cold storage. However, fruit dipped in 45 mM ascorbic acid solution resulted in about

1.23-fold higher SSC: TA ratio than 30 mM ascorbic acid-treated fruit after 28 days of cold

storage (Fig. 6.2C).


92

Fig. 6.2 Effect of different concentrations of postharvest application of ascorbic acid SSC (A), TA (B) and SSC: TA (C) in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


93


Control fruit showed quick decline in the TP and total antioxidants of peel tissues

during entire cold storage period (Fig. 6.3). Fruit treated with 45 mM ascorbic acid exhibited

about 1.56-fold higher TP, as compared to control fruit after 28 days of cold storage (Fig.

6.3A). Similarly, total antioxidants were also decreased significantly with increase in storage

period, irrespective to treatments. However, 45 mM ascorbic acid-treated fruit exhibited

about 1.30-fold higher level of total antioxidants than control fruit. Among ascorbic acid

concentrations, fruit treated with 45 mM ascorbic acid exhibited significantly about 1.06-fold

higher total antioxidants than 15 mM ascorbic acid treatment after 28 days of storage (Fig.

6.3B). Pericarp browning index exhibited negative correlation with TP and total antioxidants

in both peel (r = -0.83; r = -0.71) and pulp tissues (r = -0.81; r = -0.68), respectively (Table

6.1).

Similar to peel tissues, substantial decline in the levels of TP and total antioxidants was

also observed in pulp tissues of litchi fruit with the advancement in cold storage period (Fig.

6.4). Ascorbic acid-treated fruit (45 mM) exhibited 2.04- and 1.60-fold higher TP and total

antioxidants than control, respectively, during 28 days of cold storage. However, after 28

days of cold storage, fruit treated with 45 mM ascorbic acid showed about1.09- and 1.15-fold

higher TP and total antioxidants in pulp tissues, than 30 mM and 15 mM ascorbic acid-

treated fruit, respectively (Fig. 6.4A and B). Similarly, ascorbic acid in juice extract of litchi

pulp tissues also showed decreasing tendency during entire cold storage period. However,

decline in ascorbic acid was about 1.40-fold higher in untreated fruit after 28 days of cold

storage than 45 mM ascorbic acid-treated fruit. Among ascorbic acid concentrations, fruit

treated with 45 mM ascorbic acid resulted in 1.22-fold higher ascorbic acid in litchi fruit after

28 days of cold storage than 30 mM ascorbic acid-treated fruit (Fig. 6.4C).


94

Fig. 6.3 Effect of different concentrations of postharvest application of ascorbic acid on total phenolics (A) and total antioxidants (B) in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


95

Fig. 6.4 Effect of different concentrations of postharvest application of ascorbic acid on total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissue of litchi fruit during cold storage. Vertical bars represents ± SE of means.


96


A linear decline in the activities of SOD and CAT enzymes was observed in peel

tissues of litchi fruit during entire storage period; however, there was more rapid decline in

SOD and CAT activities in untreated fruit compared to ascorbic acid-treated fruit (Fig. 6.5A

and B). Activities of SOD and CAT enzymes were significantly about1.75- and 1.76-fold

lower in control fruit, respectively, as compared to 45 mM ascorbic acid-treated fruit after 28

days of cold storage. Fruit treated with 45 mM ascorbic acid exhibited 1.13-fold higher

activity of SOD enzyme than 15 mM ascorbic acid-treated fruit (Fig. 6.5A) and 1.25-fold

higher activity CAT enzyme than 30 mM ascorbic acid-treated fruit, after 28 days of cold

storage (Fig. 6.5B). Pericarp browning index was negatively correlated with the activities of

SOD (r = -0.81) and CAT (r = -0.65) enzymes in peel tissues of litchi fruit during cold

storage (Table 6.1). On the other hand, the activity of POD enzyme in the peel tissues

showed increasing behaviour throughout the storage period. Control fruit showed

significantly higher (1.84-fold) activity of POD enzyme than 45 mM ascorbic acid-treated

fruit after 28 days of cold storage. Application of ascorbic acid (45 mM) maintained

significantly 1.51-fold lower activity of POD enzyme than 30 mM ascorbic acid-treated fruit

after 28 days of cold storage (Fig. 6.5C). Ascorbic acid-treated fruit exhibited positive

correlation (r = 0.67) between pericarp browning index and activities of POD in peel tissues

(Table 6.1).


observed in pulp tissues of litchi fruit with advancement in cold storage period. Control fruit

showed significantly lowest activities of SOD and CAT enzymes than ascorbic acid -treated

fruit (Fig. 6.6A and B). On the other hand, after 28 days of storage, fruit treated with 45 mM

showed significantly higher activities of SOD (1.44-fold) and CAT (1.21-fold) enzymes than

30 mM ascorbic acid-treated fruit (Fig. 6.6A and B). Increasing trend was observed in the

activity of POD enzyme in pulp tissues of litchi fruit. Untreated fruit exhibited significantly

1.48-fold higher activities of POD after 28 days of cold storage, as compared to 45 mM

ascorbic acid-treated fruit. However, after 28 days of cold storage 30 mM ascorbic acid-

treated fruit exhibited 1.17-fold higher activity of POD than 45 mM ascorbic acid-treated


97

fruit (Fig. 6.6C). Activities of SOD and CAT enzymes exhibited negative correlations (r = -

0.64; -0.79) and POD enzyme showed positive correlation (r = 0.77) with pericarp browning

index in pulp tissues of ascorbic acid-treated fruit (Table 6.1).

Fig. 6.5 Effect of different concentrations of postharvest application of ascorbic acid on the activities of superoxide dismutase (A), catalase (B) and peroxidase (C) enzymes in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


98

Fig. 6.6 Effect of different concentrations of postharvest application of ascorbic acid on the activities of superoxide dismutase (A), catalase (B) and peroxidase (C) enzymes in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


99

6.5 Discussion

Red colour of the pericarp in litchi fruit was significantly affected by increase in

weight loss during cold storage. Increase in fruit weight loss could be due to constants

evaporation and transpiration processes during cold storage. These results are in compliance

with Mitra and Kar (2001) who reported increase in weight loss in ‘Bombai’ litchi fruit with

increase in storage period. Exogenous application of ascorbic acid significantly delayed

pericarp browning in litchi cv. ‘Gola’ fruit during cold storage. Previously, ascorbic acid in

combination with chitosan coating significantly reduced the rate of pericarp browning in

‘Feizixiao’ litchi and enhanced the antioxidative capacity of the fruit (Sun et al., 2010).

Similarly, anthocyanin contents were degraded by increase in browning index during

cold storage. Anthocyanin degradation has been reported to be associated with higher

activities of PPO and POD enzymes (Jiang, 2000; Zhang et al., 2005). Application of

ascorbic acid inhibited the activities of PPO and POD enzymes by enhancing the

antioxidative capacity of litchi peel and pulp tissues. These results are in accordance with

Silva et al. (2010) and Sun et al. (2010) who observed rapid loss in anthocyanin contents of

untreated fruit, as compared to litchi fruit treatment with combine application of ascorbic

acid with chitosan coating.

Activity of PPO enzyme kept on increasing during entire storage period but ascorbic

acid-treated fruit maintained significantly low activities of PPO enzymes. Similar behaviour

of PPO enzyme activity was observed when ‘Feizixiao’ litchi fruit were treated with combine

application of ascorbic acid and chitosan (Sun et al., 2010). Moisture loss triggers increase in

pH due to which activity of PPO enzyme was also increased. However, ascorbic acid dipping

might have reduced PPO activity by decreasing pH and previously Tipton and Dixon (1983)

reported that active sites of PPO enzyme are denatured at low pH. Due higher to loss of

moisture contents in control fruit, pH was increased and PPO enzyme became active. At

higher pH activity of PPO enzyme was increased in litchi cv. ‘Huaizhi’ fruit; whereas,

minimum or no activity was observed at 4.0 pH (Jiang, 2000).

SSC and TA are very important in determining the flavour and quality of litchi fruit

(Jiang and Fu, 1998). In current research, SSC of the fruit was increased during entire cold


100

storage period, irrespective of treatments applied. Similar results were also observed in

‘Bengal’ litchi, where SSC was increased when treated with hydrochloric acid (Hojo et al.,

2011). The increase in SSC was probably due to a concentration factor as water loss or to a

conversion of organic acids to glycolytic intermediates and subsequently to sugars

(Echeverria and Valich, 1989). Moisture loss probably concentrated the soluble solids of the

fruit and resulted in higher SSC as storage period progressed (Tanada-Palmu and Grosso,

2005). A substantial decline in TA was observed with the advancement of storage period and

decline of TA in control fruit was more, as compared to ascorbic acid-treated fruit. On the

other hand, SSC: TA ratio was also increased during extend cold storage period. A decline in

TA could be due to consumption of organic acids during respiration process and these results

are in accordance with Hojo et al. (2011) who reported decrease in TA of the ‘Bengal’ litchi

with increase in storage period.

TP and total antioxidants in litchi peel and pulp tissues were decreased continuously

during 28 days of storage period but exogenous application of ascorbic acid resulted in

reduced loss of TP and total antioxidants, as compared to untreated fruit. Phenolic

compounds due to their antioxidative properties are known to scavenge reactive oxygen

species (Hodges, 2003).However, higher activity of PPO has been reported to be the main

cause of decline in phenolic compounds (Baltacig et al., 2011). However, ascorbic acid

application maintained higher level of TP in fresh cut cantaloupe melon, most likely by

inhibiting PPO enzyme activities and converting o-quinones back to phenolic substrates as

reported by Lamikanra and Watson (2001). Ascorbic acid application also resulted in

increased level of total antioxidants in lettuce with correspondent increase in antioxidative

capacity (Altunkaya and Gokmen, 2008). Similarly, antioxidants are consumed during

scavenging of ROS species due to which their concentration tend to decrease during storage

(Hounsome et al., 2009). A continuous reduction in ascorbic acid was observed with increase

in storage period (Silva et al., 2010). However, ascorbic acid-treated litchi fruit maintained

higher level of ascorbic acid in their pulp tissues. Similar trend in ascorbic acid was also

reported by Silva et al. (2010) in ‘Bengal’ litchi, when treated with ascorbic acid. These

results are in line with the findings of Hojo et al. (2011) and Ray et al. (2005), where the

same behaviour of ascorbic acid of litchi fruit was observed. Oxygen stimulated conversion


101

of organic acid in sugar could be responsible for decrease in ascorbic acid during prolonged

storage period (Gimnez et al., 2003).

Fruit naturally contain SOD and CAT enzymes as a defense system against ROS

(Niranjana et al., 2009). These enzymes play major role in antioxidant defense system by

maintaining the ROS production below the threshold levels (Rao et al., 1996). In this study,

activities of SOD and CAT enzymes were decreased continuously in peel and pulp tissues of

litchi fruit. However, exogenous ascorbic acid application resulted in higher activities of

SOD and CAT enzymes, as compared to untreated fruit. Similar results were observed when

ascorbic acid treatment was applied in combination with chitosan to ‘Feizixiao’ litchi, which

increased the activities of SOD and CAT enzymes by enhancing the antioxidative capacity of

the fruit, improving the aril quality and delaying of senescence (Sun et al., 2010). On the

other hand, activities of POD in the litchi peel and pulp tissues were increased as storage

period progressed. In litchi fruit, POD hastens the oxidation of phenolics, degradation of

anthocyanin and deterioration of fruit quality in litchi (Zhang et al., 2005). However,

ascorbic acid inhibited the activity POD enzyme due to which pericarp browning was

reduced (Sun et al., 2010). These results were also supported by Mizobutsi et al. (2010) who

observed that POD enzyme activity could be inhibited more effectively at acidic pH rather

than at alkaline pH.

6.6 Conclusion

Exogenous application of 45 mM ascorbic acid prevented browning of litchi fruit for

longer period of time by maintaining higher activities of antioxidative enzymes (SOD and

CAT) along with better physico-chemical attributes in litchi cv. ‘Gola’ fruit during cold

storage.

Chapter‐7 Effect of hexanal on pericarp browning and quality of litchi fruit

102

Chapter 7

Effect of Hexanal on the Storage Life and Pericarp Browning of Litchi cv. ‘Gola’ Fruit

7.1 Abstract

Litchi is one of the most perishable sub-tropical fruit having very short shelf life and

pericarp browning is the major factor affecting its postharvest quality. This study was

conducted to investigate the effect of hexanal on pericarp browning, changes in activities of

antioxidative enzymes and biochemical quality attributes of litchi cv. ‘Gola’ fruit litchi under

cold storage conditions. Fruit harvested at physiological maturity were fumigated with

different hexanal concentrations (0, 250, 500, 750 and 1000 µL L-1) for 45 min. and then

stored at 5 ± 1°C with 90 ± 5% RH for 28 days. Physical (browning index and fruit weight

loss), biochemical quality characteristics [soluble solid concentrations (SSC), titratable

acidity (TA), SSC: TA ratio, ascorbic acid] were determined in pulp tissues; whereas,

anthocyanin contents were determined in peel tissues. On the other hand, total phenolics

(TP), total antioxidants and activities of enzymes [superoxide dismutase (SOD), catalase

(CAT), peroxidase (POD) and polyphenol oxidase (PPO)] were determined in peel and pulp

tissues of litchi fruit at 7 days interval during cold storage. Postharvest fumigation of hexanal

could not control pericarp browning and resulted in lower anthocyanin and increased weight

loss than control fruit. Maximum SSC, lowest TA and highest SSC: TA was observed in

1000 µL L-1 hexanal-treated fruit, while ascorbic acid was higher in 250 µL hexanal-treated

fruit. Control fruit exhibited higher total phenolics and total antioxidants in peel and pulp

tissues, as compared to hexanal-treated fruit. Activities of antioxidative enzymes (SOD and

CAT) and level of TP and total antioxidants in litchi peel and pulp tissues were low in

hexanal-treated fruit. On the other hand, activities of PPO and POD enzymes in litchi peel

tissues were higher in hexanal-treated fruit. Conclusively, postharvest fumigation of hexanal

could not control pericarp browning, but comparatively maintained fruit quality of ‘Gola’

litchi longer than control during cold storage.


103

7.2 Introduction

Litchi was originated near Southern China and Northern Vietnam. It is an important

commercial fruit of tropical/sub-tropical areas of the world. At present China is the largest

producer of litchi in the world (Menzel, 2001). It possesses high market value owing to its

delicious flavour of aril, vitamins and minerals, and carbohydrate (Wall, 2006).

Postharvest pericarp browning of litchi is the main postharvest problem which

involves rapid browning of skin after harvest (Kumar et al., 2013), thus affects the quality of

fruit and hinder its market potential (Rajwana et al., 2010). Various biochemical and

physiological changes for instance reduction of the membrane integrity due to lipid

peroxidation, degradation of the anthocyanin contents, increase in the activities of peroxidase

(POD), and polyphenol oxidase (PPO) and catalyzation of the super-oxidation reactions have

been reported to be connected with the browning of pericarp in litchi (Jiang, 2000).

Numerous efforts have been reported to overcome postharvest pericarp browning in litchi

with different postharvest treatments including coatings (Joas et al., 2005), application of

antioxidants (Kumar et al., 2013), oxalic acid (Zheng and Tian, 2006) and ascorbic acid (Sun

et al., 2010), however, with inconclusive results as effect of each chemical vary with

concentration, method of application, maturity stage of harvested fruit and cultivar

(Sivakumar et al., 2010). Hexanal being natural volatile compound has been reported to

increase shelf life of peach, raspberry and tomato fruit by killing the fungal spores and

reducing postharvest decay of the fruit (Song et al., 2007; Utto et al., 2008). Postharvest

hexanal application was very effective in delaying membrane degradation, thereby,

increasing the shelf of various fruit, vegetable and flowers (Paliyath and Subramanian, 2008).

Hexanal application increased the shelf life of freshly sliced apples and improved

their quality by inhibiting microbial growth during cold storage (Lanciotti et al., 1999).

Similarly, hexanal being broad spectrum fungicide increased the aroma and retarded decay in

‘Golden Delicious’ apple slices (Song et al., 1996).

As demand of the litchi fruit is increasing steadily in Pakistan and worldwide,

therefore, there is need to extend storage and shelf life of litchi fruit. At present to the best of

our knowledge no information is available regarding the effect of hexanal on pericarp


104

browning and other quality attributes of litchi. Therefore, we hypothesized that occurrence of

litchi pericarp browning could be controlled by the exogenous applications of hexanal.

Hence, current study was piloted to determine the effects of exogenous applications of

hexanal on pericarp browning, enzymatic activities and fruit quality characteristics of litchi

under cold storage conditions.


Treatments

Fruit of litchi cv. ‘Gola’ uniform size, shape and free from any physical damage were

harvested from healthy litchi trees located at Fruit Farm Nursery, Haripur, Pakistan as

described in detail in section 3.1.1 (page 34). After harvest, fruit were packed in corrugated

fiberboard boxes and were transported to Postharvest Research and Training Centre (PRTC),

Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan using

reefer van at 12oC. At arrival, fruits were immediately fumigated with hexanal for 1 h in a

sealed plastic drum containing different concentrations of hexanal (0, 250, 500, 750 or 1000

µL L-1) and then stored at 5 ± 1oC with 90 ± 5% RH for 28 days. The fruit were removed at 7

days interval from cold storage to determine fruit weight loss (%), pericarp browning index;

whereas, SSC, TA, SSC: TA ratio, ascorbic acid were determined from juice samples

obtained from pulp tissues. Moreover, TP, total antioxidants, activities of POD, CAT and

SOD enzymes were also determined at 7 days interval during cold storage in both peel as

well as pulp tissues of litchi fruit. However, activities of PPO and anthocyanin contents were

determined only from peel tissues. The study was conducted under completely randomized

design with two factors factorial arrangement (i.e. Hexanal concentrations and storage

period) having 25 fruit as an experimental unit replicated three times.

Pericarp browning

Pericarp browning was assessed by using method outlined in detail in section 3.3.1.1

(Page 36). Pericarp browning was expressed as browning index.


105

Fruit weight loss









outlined in detail in section 3.5 (Page 42) and was expressed as ∆A g-1FW.








section 3.3.2.4 (Page 38) and was expressed in mg 100 g1.










106





Statistix 10 for Windows software with two-factor factorial arrangements including hexanal

treatments and cold storage period. The effects of treatments were determined from the least


et al., 1997). Pearson’s correlations were also performed to estimate relationship between

fruit browning index, anthocyanin contents and various antioxidative enzyme activities using

Statistix 10 for Windows software (Analytical Software, Tallahasee, FL 32317, USA).

7.4 Results


Fruit weight loss was significantly increased with the progression of storage period,

irrespective to treatments. All hexanal treatments showed significantly higher weight loss

than untreated fruit. Maximum weight loss was observed in 1000 µL L-1 hexanal-treated fruit

which was 2.21-fold higher, as compared to untreated fruit. Among hexanal-treated fruit, 250

µL L-1 exhibited significantly about 1.47-fold lower fruit weight loss than 1000 µL L-1 treated

fruit after 28 days of cold storage (Fig. 7.1A).

Application of hexanal resulted in significantly higher pericarp browning index than

control fruit, due to which fruit visual appearance deteriorated. Fruit treated with 1000 µL L-1

hexanal, exhibited significantly 1.28- and 1.06-fold higher pericarp browning index than

untreated and 250 µL L-1 hexanal-treated fruit after 28 days of cold storage (Fig. 7.1B).

Anthocyanin contents in peel tissues of litchi fruit deteriorated rapidly as they were

negatively correlated (r = -0.57) with pericarp browning (Table 7.1). As pericarp browning

increased, anthocyanin contents in the litchi peel tissues of cv. ‘Gola’ were significantly

declined in all the treatments during cold storage period. All hexanal treatments exhibited

significantly higher decline in anthocyanin contents of litchi peel tissues, as compared to

control fruit. Fruit treated with 1000 µL L-1 hexanal exhibited 1.51-fold lower anthocyanin


107

contents than untreated fruit. However, among hexanal treatments, 250 µL L-1 concentration

results in higher anthocyanin contents then other treatment concentration by exhibiting 1.39-

fold higher anthocyanin contents than 1000 µL L-1 hexanal-treated fruit (Fig. 7.1C).

In litchi fruit activities of PPO enzymes were increased in all the treatments during

the cold storage period. However, control fruit exhibited 1.27-fold lower activities of PPO

enzyme in litchi peel tissue than hexanal-treated fruit. Fruit treated with 250 µL L-1 hexanal

exhibited 1.19-fold lower PPO enzyme activities than 1000 µL L-1 hexanal-treated fruit after

28 days of cold storage (Fig. 7.1D). Activities of PPO enzyme in peel tissues showed

significant positive correlation (r = 0.83) with pericarp browning index during cold storage

(Table 7.1).

Table 7.1. Relationship of pericarp browning index with activities of PPO, POD, SOD, CAT enzymes, TP and total antioxidants in hexanal-treated litchi peel and pulp tissues.

Parameter Peel Pulp

PBI vs anthocyanin contents -0.577** ---

PBI vs PPO 0.834** ---

PBI vs POD 0.695** 0.792**

PBI vs SOD -0.899** -0.893**

PBI vs CAT -0.748** -0.559**

PBI vs TP -0.827** -0.802**

PBI vs total antioxidants -0.839** -0.827**

PBI = Pericarp browning index, PPO = polyphenol oxidase, POD = peroxidase, TP = total phenolics, SOD = superoxide dismutase, CAT = catalase, ** = significant at P ≤ 0.01.


108

Fig. 7.1 Effect of different concentrations of postharvest hexanal fumigation on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and activities of polyphenol oxidase enzyme (D) in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


109

Fig. 7.2 Effect of different concentrations of postharvest hexanal fumigation SSC (A), TA (B) and SSC: TA (C) ratio in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


110

SSC, TA and SSC: TA

Increasing trend in the SSC was observed in all the treatments with the progression of

cold storage period. Control fruit exhibited significantly less increase than hexanal-treated

fruit and maintained 1.05-fold lower SSC after 28 days of cold storage. Among hexanal

concentrations, 1000 µL L-1 hexanal-treated fruit exhibited about 1.04-fold increase in SSC

than 250 µL L-1 hexanal-treated fruit after 28 days of cold storage (Fig. 7.2A). On the other

hand, TA of the fruit was declined continuously during entire cold storage period.

Decline in TA was more in hexanal-treated fruit, and 1000 µL L-1 hexanal resulted in

1.08-fold lower TA than control fruit. Among hexanal-treated fruit, lowest TA (1.18-fold)

was observed in fruit treated with 1000 µL L-1 hexanal than 250 µL L-1 hexanal after 28 days

of cold storage (Fig. 7.2B). However, with the advancement of cold storage period, linear

increase in SSC: TA ratio was observed, irrespective to treatments. Fruit treated with 1000

µL L-1 hexanal showed about 1.12- and 1.29-folds higher SSC: TA ratio, as compared to

control and 250 µL L-1 hexanal-treated fruit, respectively after 28 days of cold storage period

(Fig. 7.2C).


111

Fig. 7.3 Effect of different concentrations of postharvest hexanal fumigation on total phenolics (A) and total antioxidants (B) in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


TP and total antioxidants of litchi cv. ‘Gola’ fruit showed linear decreasing trend in

the peel tissues during entire cold storage period (Fig. 7.3). Control fruit exhibited

significantly about 1.24- and 1.08-fold higher TP and total antioxidants than hexanal-treated

fruit, respectively. Among different hexanal concentration, fruit treated with 250 µL L-1

exhibited significantly about 1.22- and 1.05-fold higher TP and total antioxidants,


112

respectively during 28 days of cold storage (Fig. 7.3A and B). Pericarp browning index

exhibited negative correlation with TP and total antioxidants in both peel (r = -0.82; r = -

0.84) and pulp tissues (r = -0.80; r = -0.82), respectively (Table 7.1). As for peel tissues,

similar trend of TP and total antioxidants was also observed in pulp tissues of litchi cv.

‘Gola’ fruit showing gradual decrease with increase in cold storage period. Control fruit

exhibited significantly 1.29- and 1.15-fold higher levels TP and total antioxidants then 1000

µL L-1 hexanal-treated fruit. However, there was no significant difference between control

fruit and 250 µL L-1 hexanal-treated fruit. Fruit treated with 250 µL L-1 hexanal exhibited

1.28-fold higher TP and 1.14-fold higher total antioxidants levels than 1000 µL L-1 hexanal-

treated fruit after 28 days of cold storage (Fig. 7.4A and B). Similarly, ascorbic acid in litchi

pulp tissues were also decreased with the progression of cold storage period. During first 14

days of cold storage, pulp tissues of control fruit exhibited significantly 1.28-fold higher

ascorbic acid than hexanal-treated fruit. However, from 14 days to 28 days of cold storage,

decline of ascorbic acid in control fruit was more than in hexanal-treated fruit. From 14 to 28

days of cold storage, fruit treated with 250 µL L-1 hexanal exhibited 1.10- and 1.21-fold

higher ascorbic acid in pulp tissues than control and 1000 µL L-1 hexanal-treated fruit,

respectively (Fig. 7.4C).


113

Fig. 7.4 Effect of different concentrations of postharvest hexanal fumigation total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


114


Activities of SOD and CAT enzymes in peel tissues of litchi fruit were significantly

decreased in all the treatments during entire storage period (Fig. 7.5A and B). Control fruit

exhibited significantly 1.53- and 1.20-fold higher activities of SOD and CAT enzymes than

1000 µL L-1 hexanal-treated fruit, respectively. Among hexanal treatments fruit, 250 µL L-1

hexanal-treated fruit exhibited about 1.51- and 1.18-fold higher activities of SOD and CAT

enzymes than 1000 µL L-1 hexanal-treated fruit after 28 days of cold storage (Fig. 7.5A and

B). Pericarp browning index was negatively correlated with the activities of SOD (r = -0.89)

and CAT (r = -0.74) enzymes in peel tissues of litchi fruit during cold storage (Table 7.1).On

the other hand, activity of POD enzyme in peel tissues of litchi cv. ‘Gola’ was increased

irrespective to treatment concentration during cold storage period. Control fruit maintained

lower activity of POD enzyme, as compared to all hexanal treatments and had 1.57-fold

lower POD activity than 1000 µL L-1 hexanal-treated fruit. Fruit treated with 250 µL L-1

hexanal exhibited 1.36-fold lower activity of POD enzyme than 1000 µL L-1 hexanal-treated

fruit after 28 days of cold storage. Hexanal-treated fruit exhibited positive correlation (r =

0.69) between pericarp browning index and activities of POD in peel tissues (Table 7.1).

Similar to peel tissues, significant decrease in the activities of SOD and CAT

enzymes were observed in pulp tissues of litchi fruit with advancement in cold storage period

(Fig. 7.6A and B). After 28 days of storage, control fruit showed significantly higher

activities of SOD (1.64-fold) and CAT (2.04-fold) enzymes than 1000 µL L-1 hexanal-

treated fruit (Fig. 7.6A and B). Increasing trend was observed in the activity of POD enzyme

in pulp tissues of litchi fruit (Fig. 7.6C). Control fruit exhibited 1.89-fold lower activity of

POD after 28 days of cold storage, as compared to 1000 µL L-1 hexanal-treated fruit. Among

different hexanal concentration, 250 µL L-1 hexanal-treated fruit exhibited significantly about

1.43-fold lower POD activity than 1000 µL L-1 hexanal-treated fruit throughout the cold

storage period (Fig. 7.6C). Activities of SOD and CAT enzymes exhibited negative

correlations (r = -0.89; -0.56) and POD enzyme showed positive correlation (r = 0.79) with

pericarp browning index in pulp tissues of hexanal-treated fruit (Table 7.1)


115

Fig. 7.5 Effect of different concentrations of postharvest hexanal fumigation on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


116

Fig. 7.6 Effect of different concentrations of postharvest hexanal fumigation on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi fruit during cold storage. Vertical bars represents ± SE of means.


117

7.5 Discussion

Significant increase in fruit weight loss was observed in the cold storage, irrespective

to treatments. Fruit weight loss mainly takes place through steady transpiration and

evaporation. Increase in weight loss with progression of storage period is in compliance with

Mitra and Kar (2001) who reported increase in weight loss in ‘Bombai’ litchi fruit. In this

study, hexanal treatments significantly increased fruit weight loss which contradicts the

findings of Song et al. (2010) who found non-significant effect of hexanal on weight loss of

blueberry fruit. Application of hexanal did not control pericarp browning in litchi cv. ‘Gola’

fruit during cold storage period. These results confirm the finding of Thavong et al. (2010)

where hexanal application was not effective in maintaining peel quality of longan fruit.

Anthocyanin contents decreased with increase in pericarp browning index during cold

storage period. Hexanal fumigation might have increased the activities of PPO and POD

enzymes, as reduction in anthocyanin contents is associated with higher activities of PPO

enzyme (Jiang, 2000). Activity of PPO enzyme kept on increasing during entire storage

period but hexanal-treated fruit exhibited significantly higher activities of PPO enzyme.

Hexanal application resulted in high moisture loss, which could have increased pH of the

fruit as high pH has been found to be associated with higher PPO enzyme activity which

consequently increase browning of pericarp tissues, as Jiang (2000) also observed increased

PPO enzyme activity at higher pH.


(Jiang and Fu, 1998). In current research, SSC of the hexanal-treated fruit was significantly

increased, as compared to control fruit during entire cold storage period. Similarly, SSC of

the tomato fruit was increased when treated with hexanal (Cheema et al., 2014). The increase

in SSC was probably due to conversion of organic acids to glycolytic intermediates and

subsequently to sugars (Echeverria and Valich, 1989). On the other hand, gradual decline in

TA of the litchi cv. ‘Gola’ fruit was observed but hexanal-treated maintained higher level of

TA after 28 days of cold storage. SSC: TA ratio was also increased significantly in all

treatments during entire cold storage period. Consumption of organic acid in sugars could be

the reason of decline in TA with increase in storage period (Hojo et al., 2011).


118

TP and total antioxidants in litchi peel and pulp tissues were decreased continuously

during 28 days of storage period but control fruit resulted in significantly higher TP and total

antioxidants levels than hexanal treatments except 250 µL L-1 hexanal-treated fruit. Phenolic

compounds play major function in scavenging reactive oxygen species under stress

conditions (Hodges, 2003), and higher activity of PPO has been reported to be the main

cause of decline in phenolic compounds (Baltacig et al., 2011). Moisture loss due to hexanal

treatment could have triggered the PPO enzyme activity causing decline in phenolics and

antioxidants level in hexanal-treated fruit. Another reason for decline in total antioxidant

might be their consumption during scavenging of ROS species under cold storage conditions

(Hounsome et al., 2009). Similarly, ascorbic acid in litchi cv. ‘Gola’ fruit also decreased in

all treatments during cold storage period. However, 250 µL L-1 hexanal-treated fruit exhibited

higher level of ascorbic acid in the pulp tissues of litchi fruit than control fruit and other

hexanal treatments. Previously, hexanal-treated tomato fruit exhibited higher ascorbic acid,

as compared to control fruit (Cheema et al., 2014). Decline in ascorbic acid could be due to

increased activity of ascorbate oxidaze enzyme (Yahia et al., 2001), which lowers the edible

quality of the fruit during storage (Jung and Watkins, 2008).

Activities of SOD and CAT enzymes were decreased continuously in peel and pulp

tissues of litchi fruit. However, control fruit exhibited overall higher activities of SOD and

CAT enzymes, as compared to hexanal-treated fruit. Fruits naturally contain antioxidative

enzymes like SOD and CAT acting as a defense system against ROS production (Niranjana

et al., 2009). SOD and CAT enzymes were directly related to the senescence of the fruit, as

their activities were decreased during storage, pericarp was turned brown and aril quality was

deteriorated (Sun et al., 2010). On the other hand, activities of POD in the litchi peel and

pulp tissues were increased significantly, irrespective of treatment, as storage period

progressed. Higher activity of POD enzymes hastens the oxidation of phenolics, degradation

of anthocyanin and deterioration of fruit quality in litchi (Zhang et al., 2005). As in present

study moisture loss was more due to hexanal application, this could have increased pH and

indirectly triggered POD enzyme activity. Previously, gradual increase in POD activity was

observed with increasing pH in pericarp of litchi cv. ‘Bengal’ as reported by Mizobutsi et al.

(2010).


119

7.6 Conclusion

Hexanal fumigation could not control browning during cold storage period. However,

among hexanal treatments, 250 µL L-1 hexanal applications showed better fruit quality

attributes than other concentration by maintaining higher activities of antioxidative enzymes

(CAT and SOD) along with lower activities of PPO and POD enzymes.

Chapter‐8 Effect of anti‐browning agents on quality of litchi cv. ‘Gola’ fruit

120

Chapter 8

Influence of Oxalic Acid, Ascorbic Acid and Hexanal on Pericarp Browning and Antioxidative Enzyme Systems in Litchi cv. ‘Gola’ Fruit

8.1 Abstract

The role of oxalic acid (OA), ascorbic acid (AA) and OA + AA + hexanal in

regulating the pericarp browning, antioxidative enzymes as well as biochemical changes in

fruit quality was investigated during cold storage of ‘Gola’ litchi fruit. Physiologically

mature fruit were immersed in an aqueous solution containing 2 mM OA, 45 mM AA plus a

combination of 2 mM OA and 45 mM AA with 250 µL L-1 hexanal using ‘Tween 20’

(0.01%) as a surfactant and stored at 5 ± 1oC with 90-95% RH. Physical (browning index and

weight loss), biochemical quality characteristics [soluble solid concentrations (SSC),

titratable acidity (TA), SSC: TA ratio, ascorbic acid] were determined in pulp juice samples;

whereas, anthocyanin contents were determined in peel tissues. On the other hand, total

phenolics (TP) and total antioxidants and activities of enzymes [superoxide dismutase

(SOD), catalase (CAT), peroxidase (POD) and polyphenol oxidase (PPO)] were determined

in peel and pulp tissues of litchi fruit at 7 days interval during cold storage. Fruit treated with

OA + AA + hexanal turned brown and quality of the fruit was also deteriorated. However,

fruit treated with 2 mM OA showed reduced weight loss, delayed pericarp browning and

maintained higher level of anthocyanin contents thorough out the storage period. SSC and

SSC: TA ratio was lower in 2 mM OA-treated fruit, while TA and ascorbic acid showed the

reverse trend. OA treatment significantly suppressed the activities of PPO and POD enzymes

in the peel tissues of litchi fruit. Total antioxidants and TP as well as activities of

antioxidative enzymes (CAT and SOD) in both peel and pulp tissues were significantly

higher in 2 mM OA-treated fruit during the entire cold storage period. Conclusively,

postharvest 2 mM OA application delayed the pericarp browning and suppressed the

activities of pericarp browning enzymes, while exhibited higher activities of antioxidative

enzymes and maintained fruit quality of ‘Gola’ litchi fruit during cold storage.


121

8.2 Introduction

Litchi being a perishable fruit crop loses its bright red skin colour soon after harvest

which reduces its market value (Kumar et al., 2013; Rajwana et al., 2010). Various factors

have been proposed to influence the occurrence and severity of pericarp browning in litchi

fruit including heat stress, senescence, chilling injuries, diseases and pests (Silva et al.,

2010). Moreover, numerous enzymes such as peroxidase (POD), polyphenol oxidase (PPO)

and ascorbic acid oxidase or reactive oxygen species including superoxide radical (O-2),

hydrogen peroxide (H2O2) and hydroxyl radicals (-OH) have been found to be involved in

pericarp browning of different fruits (Mizobutsi et al., 2010; Wang et al., 2007; Ye, 2005). In

litchi, enzymes reported to be involved in pericarp browning are PPO (De-Reuck et al., 2009;

Sun et al., 2010) and POD (Jiang, 2000; Lima et al., 2010).

Previously SO2 fumigation was used commercially to control pericarp browning in

litchi, but sulphur residues left in fruit caused harmful effects on consumer’s health

(Sivakumar et al., 2005). Therefore, other substitutes such as citric acid with cassava starch,

hydrochloric acid with PVC film, chitosan in combination with ascorbic acid and N-Acetyl

Cysteine have also been reported to delay pericarp browning in litchi (Liu et al., 2006; Silva

et al., 2010; Sun et al., 2010). Similarly, various anti-browning agents like calcium chloride,

iso-ascorbic acid, 4-hexylresorcinol and citric acid have also been found effective to control

browning in apple slices, pear slices, loquat and mango (Monsalve-Gonzalez et al., 1995;

Buta and Abbott, 2000; Ding et al., 2002; Guerrero-Beltran et al., 2005).

Earlier, postharvest application of oxalic acid (OA) has been reported to control

browning in apple and banana slices by decreasing PPO activity (Yoruk et al., 2002).

Similarly, oxalic acid-treated litchi cv. ‘Huaizhi’ fruit exhibited delayed pericarp browning

when kept at ambient conditions (Zheng and Tian, 2006). Similarly, ascorbic acid (AA) was

also reported to prevent browning and delay senescence due to its antioxidative capabilities

in various fresh cut fruits and vegetables (Suttirak and Manurakchinakorn, 2010). Postharvest

application of AA delayed pericarp browning of litchi fruit by maintaining membrane

integrity and inhibited the leakage rate through suppressing the activities of PPO and POD


122

enzymes (Sun et al., 2010) or the formation ascorbyl which directly scavenges ROS

(Yamaguchi et al., 1999) and reduces the o-quinones produced by PPO (Robert et al., 2003).

Hexanal is another volatile compound which has been reported to increase shelf life of peach,

rasp berry and tomato fruit by killing the fungal spores and reducing postharvest decay of the

fruit (Song et al., 2007; Utto et al., 2008). Hexanal application effectively delayed membrane

degradation, thereby, increased the postharvest shelf life of various fruit, vegetables and

flowers (Paliyath and Subramanian, 2008). Moreover, application of hexanal has also been

reported to enhance the aroma and shelf life of apple slices (Song et al., 1996), as shelf life of

freshly cut apple slices was significantly increased by hexanal application under cold storage

(Lanciotti et al., 1999).

It appears that application of oxalic acid (Chapter 5) and ascorbic acid (Chapter 6)

can effectively control the pericarp browning; however, to the best of our knowledge their

combine application with hexanal on pericarp browning under prolonged cold storage on

litchi cv. ‘Gola’ fruit have not been investigated. Therefore, present study was conducted to

further explore the anti-browning characteristics of oxalic acid, ascorbic alone and in

combination with hexanal to select the best chemical concentration to delay pericarp

browning in commercial litchi cultivar ‘Gola’ under extended cold storage conditions.


Treatments

Fruit of litchi cv. ‘Gola’ of uniform in size, shape and free from any physical damage

were harvested from Fruit Farm Nursery, Haripur, KPK, Pakistan. After harvest, fruit were

packed in corrugated fiberboard boxes and were transported to Postharvest Research and

Training Centre (PRTC), Institute of Horticultural Sciences, University of Agriculture,

Faisalabad, Pakistan using reefer van at 12oC as mentioned in detail in section 3.1.1 (Page

34). Fruit were treated with aqueous solution of 2 mM OA, 45 mM AA and their

combination (OA + AA) using ‘Tween-20’ (0.01%) as a surfactant. Fruit treated with

combine solution of OA and AA were further fumigated with 250 µL L-1 hexanal. After

application of treatments fruit were stored at 5 ± 1oC with 90 ± 5% RH for 28 days. Fruit


123

were removed at 7 days interval from cold storage to determine fruit weight loss (%),

pericarp browning index; whereas SSC, TA, SSC: TA ratio, ascorbic acid were determined

from juice samples obtained from pulp tissues. Moreover, TP, total antioxidants, activities of

POD, CAT and SOD enzymes were also determined at 7 days interval during cold storage in

both peel as well as pulp tissues of litchi fruit. However, activity of PPO and anthocyanin

contents was determined only from peel tissues. The study was conducted under completely

randomized design with two factors factorial arrangement (i.e. treatments and storage period)

having 25 fruit as an experimental unit replicated three times.

Pericarp browning

Pericarp browning was assessed by using the method outlined in detail in section


Fruit weight loss

Fruit weight loss was calculated by digital balance as described detail in section
















124






measured as U mg protein-1 (section 3.6.1; page 45), where one unit was defined as “an









Statistix 10 for Windows software with two factor factorial arrangements including treatment

concentrations and storage period. Each experimental unit was consisted of 25 fruits with

three replicates. The effects of treatments were determined from the least significant

differences test (Fisher’s LSD) at P ≤ 0.05, where the F test was significant (Steel et al.,

1997).

8.4 Results


Fruit weight loss was significantly increased in ‘Gola’ litchi fruit during the entire

storage period, irrespective to treatments (Fig. 8.1A). However, treated fruit showed less

weight loss as compared to control. Among the treatments, lowest fruit weight loss (8.90%)

was observed in fruit treated with 2 mM OA, as compared to control (12.86%) after 28 days

of cold storage. Pericarp browning index was also increased, irrespective to treatments with

the progression of storage period. Among different treatments, application of OA (2 mM)

and AA (45 mM) were the most effectives in delaying pericarp browning. After 28 days of


125

cold storage, the pericarp browning index was about 1.42-, 1.33- and 1.12-fold higher in fruit

treated with 2 mM OA + 45 mM AA + 250 µL L-1 hexanal, control and AA-treated fruit,

respectively, as compared to 2 mM OA-treated fruit (Fig. 8.1B). As the storage period

progressed, anthocyanin content in the peel tissues of litchi cv. ‘Gola’ declined continuously

in both treated and control fruit. Fruit subjected to 2 mM OA or 45 mM AA exhibited higher

anthocyanin contents in the peel tissues of ‘Gola’ litchi as compare to other treatments.

Among the treatments, application of 2 mM OA-treated fruit resulted in significantly highest

average anthocyanin contents (1.67 ∆g-1FW) compares to all others treatments and was 1.6-

fold higher than 2 mM OA + 45 mM AA + 250 µL L-1 hexanal-treated fruit after 28 days of

cold storage (Fig. 8.1C). Irrespective to the treatments, ‘Gola’ litchi fruit showed a

continuous and gradual increase in the activity of PPO enzymes in the peel tissues with the

progression of storage period. This increase in PPO activity was more noticeable in control

and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal treatments, as compared to 2 mM OA or

45 mM AA application. After 28 days of cold storage period, PPO enzyme activity in the

peel tissues of 2 mM OA- and 45 mM AA-treated litchi fruit was 1.3- and 1.1-folds lower

than control (Fig. 8.1D).


126

Fig. 8.1 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase enzyme (D) in peel tissues of litchi cv. ‘Gola’ fruit during cold storage. Vertical bars represents ± SE of means.


127

Fig. 8.2 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal on SSC (A), TA (B) and SSC: TA (C) ratio in pulp tissues of litchi cv. ‘Gola’ fruit during cold storage. Vertical bars represents ± SE of means.


128

SSC, TA and SSC: TA ratio

A significant linear increasing trend was observed for SSC contents in litchi cv. ‘Gola’ fruit

subjected to all treatments during 28 days of cold storage period. Mean SSC contents were

about 1.04-fold higher in treated than in control fruit. The highest level of SSC contents were

observed after 28 days of cold storage and about 1.05-fold higher in treated, as compared to

control fruit (Fig. 8.2A). TA substantially declined, irrespective to treatments with the

progression of cold storage period. All treated fruit showed significantly higher TA during

cold storage in contrast to the control fruit. Among the different treatments, application of 2

mM OA resulted in significantly highest TA (0.30 %) compares to all other treatments after

28 days of storage period (Fig. 8.2B). A significant increase in SSC: TA ratio was also

observed during storage of litchi cv. ‘Gola’ fruit, irrespective to treatments. After 28 days of

cold storage, SSC: TA ratio was about 2.97-, 1.28-, 1.46- and 1.36- fold higher in 2 mM OA,

45 mM AA and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal-treated fruit, respectively, as

compared to untreated fruit (Fig. 8.2C).


TP and antioxidants of the peel tissues of litchi fruit decreased significantly during

entire cold storage period. Pre-storage application of 2 mM OA and 45 mM AA prevented

the reduction in TP and total antioxidant, as compared to other treatments during cold storage

(Fig. 8.3A and B). The mean TP and total antioxidant contents after 28 days of cold storage

were significantly higher (155.25 mg GAE 100 g-1 and 41.94 %inhibition) in 2 mM OA-

treated fruit which was 1.3- and 1.4-fold higher than 2 mM OA + 45 mM AA + 250 µL L-1

hexanal treatment (122.52 mg GAE 100 g-1 and 28.95 %inhibition), respectively.

Significant linear decline in the TP, total antioxidant ascorbic acid was observed in

the pulp tissues of ‘Gola’ litchi fruit with the advancement of cold of storage period (Fig.

8.4). However, decrease in TP and total antioxidant contents during cold storage period was

more pronounced in control than in 2 mM OA or 45 mM AA-treated fruit. On the other hand,

application of 2 mM OA + 45 mM AA + 250 µL L-1 hexanal resulted in less TP and total

antioxidants throughout the storage period. After 28 days of cold storage, TP and total


129

antioxidants were significantly 1.17- and 1.19-folds higher in pulp tissues of 2 mM OA-

treated fruit (Fig. 8.4A and B). Gradual decline in ascorbic acid was observed in litchi pulp

tissues throughout the cold storage period. Among different treatments, fruit treated with 45

mM ascorbic acid exhibited 1.34-fold higher ascorbic acid after 28 days of cold storage.

However, the mean ascorbic acid was significantly 1.18-fold higher in 2 mM OA-treated

fruit than in control (Fig. 8.4C).

Fig. 8.3 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal on total phenolics (A) and total antioxidants (B) in peel tissues of litchi cv. ‘Gola’ fruit during cold storage. Vertical bars represents ± SE of means.


130

Fig. 8.4 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal on total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi cv. ‘Gola’ fruit during cold storage. Vertical bars represents ± SE of means.


131


Activities of SOD and CAT enzyme in the peel tissues was significantly decreased

during prolonged cold storage period in litchi cv. ‘Gola’ fruit, irrespective to treatments.

Exogenous application of 2 mM OA and 45 mM AA significantly suppressed the decrease in

activities of SOD and CAT enzyme in the peel tissues during cold storage period (Fig. 8.5A

and B). After 28 days of cold storage, 2 mM OA and 45 mM AA-treated fruit exhibited 1.23-

and 1.16-fold higher SOD activity in the peel tissues, as compared to control (Fig. 8.5A).

CAT activity in the peel tissue of 2 mM OA-treated fruit was 1.29-fold higher than in control

(Fig. 8.5B). On the other hand, activity of POD enzyme significantly increased in litchi peel

tissues as the storage period progressed. This increase in POD activity was higher in control

fruit and fruit treated with combined application of 2 mM OA + 45 mM AA + 250 µL L-1

hexanal, as compared to individual application of 2 mM OA and 45 mM AA. However, fruit

treated with 2 mM OA and 45 mM AA showed about 1.16- and 1.07-folds lower activity of

POD enzyme after 28 days of cold storage than control (Fig. 8.5C). Activities of SOD and

CAT enzymes were also decreased in the pulp tissues of litchi during cold storage period.

Exogenous application of 2 mM OA or 45 mM AA alone exhibited higher activities of SOD

and CAT enzymes in the pulp tissues during the entire cold storage period (Fig. 8.6A and B).

Alone application of 2 mM OA or 45 mM AA significantly delayed this decline in the SOD

activity in the pulp tissues, as compared to control.

The mean SOD activity during cold storage was significantly 1.43- and 1.16-folds

higher in 2 mM OA- and 45 mM AA-treated fruit, respectively, as compared to control fruit

(Fig. 8.6A). Fruit treated with 2 mM also showed 1.14-fold higher CAT enzyme activity, as

compared to control after 28 days of cold storage (Fig. 8.6B). Like in peel tissues, activity of

POD enzyme was also increased in pulp tissues of litchi cv. ‘Gola’ fruit with the

advancement of cold storage period. Fruit subjected to exogenous 2 mM OA or 45 mM AA

treatment exhibited lower activity of POD enzyme in the pulp tissue of litchi throughout the

storage period, as compared to other treatments. Peak activity of POD enzyme was observed

after 28 days of cold storage which was significantly 1.25- and 1.14-fold lower in 2 mM OA-

and 45 mM AA-treated fruit than control, respectively (Fig. 8.6C).


132

Fig. 8.5 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues of litchi cv. ‘Gola’ fruit during cold storage. Vertical bars represents ± SE of means.

(A)


133

Fig. 8.6 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi cv. ‘Gola’ fruit during cold storage. Vertical bars represents ± SE of means.


134

8.5 Discussion

As expected, fruit weight loss was increased in all the treatments with the progression

of cold storage period but 2 mM OA treatment resulted in reduced weight loss, as compared

to other treatments. Major reason of weight loss could be the metabolic activities and

transpiration taking place during period of storage (Narayana et al., 1996). Increase in fruit

weight loss with progression of storage period was also observed by Mitra and Kar (2001) in

‘Bombai’ litchi fruit. Oxalic acid-treated pomegranate fruit showed less fruit weight loss

during prolonged cold storage period (Sayyari et al., 2010). Pericarp browning index was

also increased irrespective of treatments during cold storage period but 2 mM OA showed

lower pericarp browning followed by 45 mM AA-treated fruit. In litchi, higher activities of

PPO and POD enzymes are considered to cause pericarp browning (Jiang et al., 2006;

Underhill and Critchley, 1995). Increase in pericarp browning due to higher weight loss was

also reported by Joas et al. (2005) in ‘Kwai Mi’ litchi. However, OA-treated fruit exhibited

less pericarp browning and high anthocyanin contents than control fruit during cold storage

period. Previous reports indicated that, exogenous application of OA in ‘Huaizhi’ litchi

(Zheng and Tian, 2006) and AA application in ‘Feizixiao’ litchi resulted in lower pericarp

browning (Sun et al., 2010). On the other hand, 2 mM OA + 45 mM AA+ 250 µL L-1

hexanal treatment could not control pericarp browning. Hexanal could have masked the anti-

browning properties of OA and AA due to which fruit turned brown. These results confirm

the findings of Thavong et al. (2010) who observed reduced acceptability of longan peel due

to hexanal treatment. As browning increased, reduction in anthocyanin contents of litchi

pericarp was observed. However, 2 mM OA maintained higher anthocyanin contents

followed by 45 mM AA, as compared to untreated fruit and 2 mM OA + 45 mM AA+ 250

µL L-1 hexanal. Exogenous application of 2 mM OA and 45mM AA exhibited higher

anthocyanin contents in litchi pericarp by preserving oxidized material in litchi fruit during

storage (Zheng and Tian, 2006; Sun et al., 2010). With the degradation of anthocyanin and

increased pericarp browning, activity of PPO enzyme in pulp tissues of litchi fruit was also

increased during cold storage period. However, activity of PPO enzyme was significantly

low in fruit treated with 2 mM OA followed by 45 mM AA, as compared to other treatments.


135

Dipping of fruit in acidic solution caused decrease in pH due to which PPO activity was

retarded in ’Huaizhi’ litchi fruit (Jiang, 2000) as active sites of PPO were denatured at low

pH (Tipton and Dixon, 1983).

SSC of the litchi pulp tissues was increased in all the treatments during entire period

of cold storage. However, 2 mM OA and 45 mM AA applications maintained lower values of

SSC, as compared to other treatments. OA (2 mM) and AA (45 mM) treated fruit reduced

moisture loss by improving membrane integrity and that’s why SSC of the fruit remained

stable. Increase in SSC with the progression of storage period was also reported in ‘Bengal’,

‘Bedana’ and ‘Shahi’ litchi fruit (Hojo et al., 2011; Aklimuzzaman et al., 2011; Rajak et al.,

2014). Similar observation in SSC was also reported by Tanada-Palmu and Grosso (2005)

who reported that moisture loss was the major reason of increase in SSC. On the other hand,

TA of ‘Gola’ litchi pulp tissues was decreased throughout the cold storage period. However,

2 mM OA-treated fruit exhibited significantly higher TA, as compared to all other

treatments. Earlier, similar trend in TA evolution during cold storage was observed in OA-

treated mango fruit (Zheng et al., 2007a). Hydrochloric acid-treated litchi fruit also showed

decline in TA due to consumption of organic acids in respiration process (Hojo et al., 2011).

Continuous decline in TP and total antioxidants of litchi peel and pulp tissues was

observed throughout the cold storage period, but 2 mM OA-treated fruit maintained

significantly higher levels of TP and total antioxidants followed by 45 mM AA treatment, as

compared to other treatments. Higher PPO enzyme activity has been reported to be the major

cause of decline in phenolic compounds (Baltacig et al., 2011). Previously, 2.5 mM AA-

treated fresh cut cantaloupe melon exhibited higher TP by reducing the activity of PPO

enzyme and converting o-quinones back to phenolic substrates (Lamikanra and Watson,

2001). On the other hand, postharvest OA application increased the antioxidative potential of

pomegranate fruit stored for 84 days at 2°C (Sayyari et al., 2010). Total antioxidants are

consumed in scavenging of ROS under storage conditions (Hounsome et al., 2009). AA

contents of litchi pulp tissues were also decreased with increase storage period and these

results support the findings of Hojo et al. (2011) who reported decrease in AA contents of

‘Bengal’ litchi fruit with the advancement of cold storage period. Exogenous application of 2


136

mM OA exhibited significantly higher AA contents than all other treatments. Previously,

ascorbic acidic treatment maintained higher AA contents in ‘Bengal’ litchi fruit (Silva et al.,

2010).

Substantial decline in the activities of SOD and CAT enzymes was observed in peel

and pulp tissues of litchi cv. ‘Gola’ with the enhancement of cold storage period. However, 2

mM OA treatment maintained higher activities of these enzymes, as compared to other

treatments. SOD and CAT enzymes are believed to play important role in protecting

antioxidants against ROS (Niranjana et al., 2009). OA application protected litchi fruit from

oxidative stress by maintaining higher activities of CAT and SOD enzymes, thereby,

decreased pericarp browning index (Zheng and Tian, 2006). Previously, OA application

resulted in higher activities of SOD and CAT enzymes in peach fruit kept at ambient

conditions (Zheng et al., 2007b). Meanwhile, increased activities of POD enzyme in peel and

pulp tissues were observed in peel and pulp tissues of the litchi cv. ‘Gola’ fruit with the

enhancement of cold storage period. However, activity of POD enzyme was low in 2 mM

OA-treated fruit both in peel and pulp tissues. Previously, Zheng and Tian (2006) observed

lower POD enzyme activities in OA-treated litchi fruit, as compared to untreated fruit.

8.6 Conclusion

Combination of hexanal with OA and AA could not control pericarp browning and

quality of litchi cv. ‘Gola’ fruit. Among all the treatments, application of 2 mM OA was

found superior in retaining the red colour for longer period of time probably by maintaining

higher activities of antioxidative enzyme (SOD and CAT) and better fruit quality attributes in

litchi cv. ‘Gola’ fruit during entire cold storage period.

Chapter‐9 Effect of anti‐browning agents on quality of litchi cv. ‘Bedana’ fruit

137

Chapter 9

Influence of Exogenous Application of Oxalic Acid, Ascorbic Acid and their Combination with Hexanal Fumigation on Pericarp Browning and

Quality of Litchi cv. ‘Bedana’ Fruit

9.1 Abstract

Litchi fruit loses its visual appearance and market value due to pericarp browning

with in few hour after harvest. Therefore, present study was conducted to investigate the

influence of oxalic acid (OA), ascorbic acid (AA) and OA + AA + Hexanal on pericarp

browning and changes in the biochemical quality attributes in litchi cv. ‘Bedana’ fruit. Fruit

harvested at physiological maturity were dipped in chemical concentrations standardized

from previous experiments (2 mM OA, 45 mM AA individually and in combination with 250

µL L-1 hexanal from chapter 5, 6 and 7) with tween 20 (0.01%) as a surfactant and stored for

28 days at 5 ± 1oC with 90-95% RH. Physical (browning index and weight loss), biochemical

quality characteristics [soluble solid concentrations (SSC), titratable acidity (TA), SSC: TA

ratio, ascorbic acid] were determined pulp juice samples, whereas, anthocyanin contents were

determined in peel tissues. On the other hand, total phenolics (TP) and total antioxidants and

activities of enzymes [superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and

polyphenol oxidase (PPO)] were determined in peel and pulp tissues of litchi fruit at 7 days

interval during cold storage. Postharvest application of 2 mM OA reduced fruit weight loss

and delayed pericarp browning by maintaining higher anthocyanin contents, as compared to

other treatments. TA and ascorbic acid were higher in fruit treated with 2 mM OA, as

compared to other treatments. Activities of PPO and POD enzymes in litchi peel tissues were

reduced in fruit treated with 2 mM OA during entire cold storage period. Activities of

antioxidative enzymes (SOD and CAT) and level of TP and total antioxidants in litchi peel as

well as pulp tissues were higher in 2 mM OA-treated fruit. In conclusion, postharvest

application of 2 mM OA significantly delayed pericarp browning and maintained better

quality of ‘Bedana’ litchi fruit during cold storage.


138

9.2 Introduction

Litchi belongs to the tropical and subtropical areas and is famous for its delicious

taste (Aklimuzzaman et al., 2011). It has a bright-red pericarp and a translucent pulp of great

nutritional value (Salomao et al., 2006). However, litchi being a perishable fruit has very

short shelf life due to the fact that its pericarp turns brown within 72 h after harvest (Del-

Aguila et al., 2009).

Pericarp browning in litchi fruit has been attributed to desiccation by temperature

stress, chilling injuries, pests and diseases (Scott et al., 1982; Fitzel and Coats, 1995).

Browning is accompanied by several changes that commonly results from the degradation of

the anthocyanin pigments by different enzymes particularly polyphenol oxidase (PPO),

peroxidase (POD) and ascorbic acid (Mizobutsi et al., 2010) or by free radicals such as

superoxide (O-2), hydrogen peroxide (H2O2) and hydroxyl radicals (-OH) known as reactive

oxygen species (ROS) (Wang et al., 2007; Ye, 2005). ROS production is in part due to failure

of antioxidative enzyme system which eliminates these free radicals (Kanazawa et al., 2000).

Excessive production of ROS can damage proteins, lipids and consequently leads to the loss

of membrane integrity and functionality (Yang et al., 2008). However, pericarp browning can

be delayed by using chemicals having anti-browning properties. Earlier, sulfur dioxide

fumigation (Ducamp-Collin et al., 2008;), hydrochloric acid (Jiang et al. 2004), citric acid

(Terdbaramee et al., 2003) and ascorbic acid (Silva et al. 2010) in combination with different

edible coatings have been used commercially to control pericarp browning (Zhang and

Quantick, 1997; Sun et al., 2010).

Previously, AA has been reported to prevent pericarp browning and delay senescence

in various fresh cut fruit and vegetable (Suttirak and Manurakchinakorn, 2010) by increasing

membrane integrity and reducing membrane leakage rate (Sun et al., 2010) or by the

formation ascorbyl which directly scavenges ROS (Yamaguchi et al., 1999) and reduces the

o-quinones produced by PPO (Robert et al., 2003). Similarly, OA application has also been

found to control pericarp browning in apple, banana (Yoruk et al., 2002) and ‘Huaizhi’ litchi

at ambient conditions (Zheng and Tian, 2006). On the other hand, hexanal being natural


139

volatile compound in plants has anti-microbial characteristics on the fruit kept at low

temperatures (Utto et al., 2008).

The results of the previous experiments reveals that application of oxalic acid and

ascorbic acid effectively controlled pericarp browning but hexanal application could not

provide satisfactory results. As combine application of OA and AA with had not been tested

on litchi fruit. Therefore, to confirm the results of previous experiment (Chapter 8), we

hypothesize that 2 mM, 45 mM and their combination 250 µL L-1 can be effective in

controlling pericarp browning and improving quality of another commercial litchi cv.

‘Bedana’ under extended storage conditions.


Treatments

Fruit of litchi cv. ‘Bedana’ uniform in size, shape and free from any physical damage

were harvested from the Fruit Farm Nursery, Haripur KPK, Pakistan. After harvest, fruit

were packed in corrugated fiberboard boxes and were transported to Postharvest Research

and Training Centre (PRTC), Institute of Horticultural Sciences, University of Agriculture,

Faisalabad, Pakistan using reefer van at 12oC (Section 3.1.1; page 34). Fruit were treated

with aqueous solution of 2 mM OA, 45 mM AA and their combination (OA + AA) using

‘Tween-20’ (0.01%) as a surfactant. Fruit treated with combine solution of OA and AA were

further fumigated with 250 µL L-1 hexanal. After application of treatments fruit were stored

at 5 ± 1oC with 90 ± 5% RH for 28 days. Fruit were removed at 7 days interval from cold

storage to determine fruit weight loss (%), pericarp browning index; whereas SSC, TA, SSC:

TA ratio, ascorbic acid were determined from juice samples obtained from pulp tissues.

Moreover, TP, total antioxidants, activities of POD, CAT and SOD enzymes were also

determined at 7 days interval during cold storage in both peel as well as pulp tissues of litchi

fruit. However, activity of PPO and anthocyanin contents was determined only from peel

tissues. The study was conducted under completely randomized design with two factors

factorial arrangement (i.e. treatments and storage period) having 25 fruit as an experimental

unit replicated three times.


140

Pericarp browning

Pericarp browning was determined by using the method outlined in detail in section


Fruit weight loss





















measured as U mg protein-1 (section 3.6.1; page 45), where one unit was defined as “an




141







Statistix 10 for Windows software with two factor factorial arrangements including

treatments and storage period. Each experimental unit was consisted of 25 fruits with three

replicates. The effects of treatments were determined from the least significant differences

test (Fisher’s LSD) at P ≤ 0.05, where the F test was significant (Steel et al., 1997).

9.4 Results


During cold storage period, irrespective to treatments, all fruit exhibited significant

increase in fruit weight loss (Fig. 9.1A). However, fruit treated with 2 mM OA showed

significantly less fruit weight loss, as compared to control, 45 mM AA and 2 mM OA + 45

mM AA + 250 µL L-1 hexanal-treated fruit during entire storage period. Fruit treated with 2

mM OA + 45 mM AA + 250 µL L-1 hexanal resulted in about 1.95-fold higher fruit weight

loss than fruit treated with 2 mM OA (Fig. 9.1A). Pericarp browning also increased

significantly in all treatment with increase in cold storage period. Fruit treated with 2 mM

OA exhibited significantly lower pericarp browning index in contrast to all other treatments.

Maximum pericarp browning index was observed in fruit treated with 2 mM OA + 45 mM

AA + 250 µL L-1 hexanal which was 1.34-fold higher than fruit treated with 2 mM OA

during entire cold storage period (Fig. 9.1B). On the other hand, anthocyanin contents

showed significant gradual decline with the advancement of cold storage period. Fruit treated

with 2 mM OA exhibited higher anthocyanin contents than all other treatments. Fruit treated

with 2 mM OA + 45 mM AA + 250 µL L-1 hexanal resulted in significantly lower (1.27-fold)

anthocyanin contents, as compared to 2 mM OA-treated fruit during entire cold storage

period (Fig. 9.1C). Similarly, activity of PPO enzyme in litchi peel tissues were increased


142

gradually with the advancement of cold storage period regardless to treatments applied. Fruit

treated with 2 mM OA exhibited significantly less PPO enzyme activity than other

treatments. Meanwhile, untreated fruit showed 1.35-fold higher PPO enzyme activity, as

compared to 2 mM OA-treated fruit (Fig. 9.1D).

SSC, TA and SSC: TA

SSC increased gradually in all the treatments with increase in cold storage period.

Significant differences in SSC were observed in all the treatments; however, untreated fruit

exhibited higher SSC than all the treatments throughout the cold storage duration. Control

fruit showed significantly about 1.09-fold higher SSC, as compared 2 mM OA + 45 mM AA

+ 250 µL L-1 hexanal-treated fruit (Fig. 9.2A). On the other hand, TA of the fruit was

decreased continuously with the progression of cold storage period. Control fruit and 2 mM

OA + 45 mM AA + 250 µL L-1 hexanal-treated fruit did not show significant differences

regarding decline in TA. However, fruit treated with 2 mM OA exhibited significantly 1.78-

fold higher TA than fruit treated with 2 mM OA + 45 mM AA + 250 µL L-1 hexanal (Fig.

9.2B). SSC: TA ratio was increased significantly with advancement of cold storage period.

Control fruit resulted in significantly 1.54-fold higher SSC: TA ratio than 2 mM OA-treated

fruit. However, no significant differences were observed between control and 2 mM OA + 45

mM AA + 250 µL L-1 hexanal-treated fruit regarding SSC: TA ratio (Fig. 9.2C).


143

Fig. 9.1 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA+ 45 mM AA+ 250 µL L-1 hexanal on fruit weight loss (A), pericarp browning (B), anthocyanin contents (C) and polyphenol oxidase enzyme (D) in peel tissues of litchi cv. ‘Bedana’ fruit during cold storage. Vertical bars represents ± SE of means.


144

Fig. 9.2 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA+ 45 mM AA+ 250 µL L-1 hexanal on SSC (A), TA (B) and SSC: TA (C) ratio in pulp tissues of litchi cv. ‘Bedana’ fruit during cold storage. Vertical bars represents ± SE of means.


145


TP and total antioxidants level were decreased in peel tissues of litchi cv. ‘Bedana’

fruit, irrespective to treatments with increase in cold storage period (Fig. 9.3). Fruit treated

with 2 mM OA exhibited significantly 1.17-fold higher TP, as compared to fruit treated with

2 mM OA + 45 mM AA + 250 µL L-1 hexanal (Fig. 9.3A). Similarly, total antioxidants in

peel tissues were decreased significantly with increase in storage period, irrespective to

treatment. However, fruit treated with 2 mM OA exhibited significantly higher level of total

antioxidants than all other treatments. Fruit treated with 2 mM OA resulted in 1.26-fold

higher level of total antioxidants than fruit treated with 2 mM OA + 45 mM AA + 250 µL L-1

hexanal (Fig. 9.3B).

Similar to peel tissues, level of TP and total antioxidants was also decreased in pulp

tissues of litchi cv. ‘Bedana’ fruit with the advancement of cold storage period (Fig. 9.4A and

B). Fruit treated with 2 mM OA exhibited 1.15- and 1.16-fold higher TP and total

antioxidants in pulp tissues, as compared to 2 mM OA + 45 mM AA + 250 µL L-1 hexanal-

treated fruit (Fig. 9.4A and B). Ascorbic acid in pulp tissues also showed continuous decline

during entire storage period. However, fruit treated with 2 mM OA exhibited 1.20-fold

higher ascorbic acid than fruit treated with 2 mM OA + 45 mM AA + 250 µL L-1 hexanal

(Fig. 9.4C).


A linear decline in the activities of SOD and CAT enzymes in peel tissues of litchi cv.

‘Bedana’ fruit was observed during entire storage period; however, fruit treated with 2 mM

OA maintained higher activities of these enzymes. As activities of SOD and CAT enzymes

were about 1.32- and 1.28-folds higher in fruit treated with 2 mM OA, as compared to 2 mM

OA + 45 mM AA + 250 µL L-1 hexanal (Fig. 9.5A and B). On the other hand, increasing

trend in the activities POD enzyme was observed during entire cold storage. Fruit treated

with 2 mM OA maintained significantly lower (1.33-fold) activity of POD enzymes in peel

tissues of litchi, as compared to 2 mM OA + 45 mM AA + 250 µL L-1 hexanal (Fig. 9.5C).


146


observed in pulp tissues of litchi cv. ‘Bedana’ fruit with advancement in cold storage period.

Although, fruit treated with 2 mM OA exhibited significantly higher activities of these

enzymes, than all other treatments. Activities of SOD and CAT enzymes were about 1.27-

and 1.18-folds higher in fruit treated 2 mM OA, as compared to 2 mM OA + 45 mM AA +

250 µL L-1 hexanal (Fig. 9.6A and B). Increasing trend in the activity of POD enzyme in

litchi pulp tissues was observed during entire cold storage period. Though, activity of POD

enzyme was significantly low (1.24-fold) in 2 mM OA-treated fruit, as compared to fruit

treated with 2 mM OA + 45 mM AA + 250 µL L-1 hexanal (Fig. 9.6C).

Fig. 9.3 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA+ 45 mM AA+ 250 µL L-1 hexanal on total phenolics (A) and total antioxidants (B) in peel tissues of litchi cv. ‘Bedana’ fruit during cold storage. Vertical bars represents ± SE of means.


147

Fig. 9.4 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA+ 45 mM AA+ 250 µL L-1 hexanal on total phenolics (A), total antioxidants (B) and ascorbic acid (C) in pulp tissues of litchi cv. ‘Bedana’ fruit during cold storage. Vertical bars represents ± SE of means.


148

Fig. 9.5 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA+ 45 mM AA+ 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in peel tissues of litchi cv. ‘Bedana’ fruit during cold storage. Vertical bars represents ± SE of means.


149

Fig. 9.6 Effect of 2 mM oxalic acid (OA), 45 mM ascorbic acid (AA) and 2 mM OA+ 45 mM AA+ 250 µL L-1 hexanal on activities of superoxide dismutase (A), catalase (B) and peroxidase enzymes (C) in pulp tissues of litchi cv. ‘Bedana’ fruit during cold storage. Vertical bars represents ± SE of means.


150

9.5 Discussion

Increase in fruit weight loss was observed with the advancement of cold storage

period. However, fruit treated with 2 mM OA and 45 mM AA resulted in reduced fruit

weight loss, as compared to the control and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal.

Weight loss takes place due to moisture loss through transpiration, respiration and various

metabolic activities taking place in fruit with progression of cold storage period (Narayana et

al., 1996). Increase in fruit weight loss with progression of storage period is in compliance

with Mitra and Kar (2001); as they also reported increase in weight loss in ‘Bombai’ litchi

fruit. These results confirms the findings of Sayyari et al. (2010), who observed less fruit

weight loss due to 6 mM OA application in pomegranate fruit, when stored at 2°C for 84

days. Due to progressive weight loss, pericarp browning was also increased during cold

storage period. Increase in pericarp browning due to higher weight loss was also reported by

Joas et al. (2005) in ‘Kwai Mi’ litchi. However, OA-treated fruit exhibited less pericarp

browning and high anthocyanin contents than control fruit during cold storage period.

Similarly, exogenous application of 2 and 4 mM OA resulted in lower pericarp browning

index and higher anthocyanin contents in ‘Huaizhi’ litchi when kept at ambient conditions

(Zheng and Tian, 2006). Similarly, exogenous 40 mM AA application resulted in reduced

pericarp browning in ‘Feizixiao’ litchi (Sun et al., 2010). On the other hand, 2 mM OA + 45

mM AA + 250 µL L-1 hexanal could not control pericarp browning and these results can be

correlated with the study conducted by Thavong et al. (2010) who observed reduced

acceptability of longan peel due to hexanal treatment. As browning increased, reduction in

anthocyanin contents of litchi pericarp was observed. However, 2 mM OA application

maintained higher anthocyanin contents followed by 45 mM AA, as compared to untreated

fruit and 2 mM OA + 45 mM AA + 250 µL L-1 hexanal-treated fruit. OA being a natural

antioxidant might have preserved anthocyanin contents from degradation and conversion into

brown colour by-products as it plays a significant role in preserving oxidized material (Zheng

and Tian, 2006). Similarly, application of 45 mM AA also maintained higher anthocyanin

level than control fruit. These results confirms the findings of Silva et al. (2010) and Sun et

al. (2010) who observed rapid loss in anthocyanin contents of untreated fruit, as compared to

AA-treated litchi fruit. With the degradation of anthocyanin and increased pericarp


151

browning, activities of PPO and POD enzymes were also increased during cold storage

period. However, activities of PPO and POD enzymes were significantly low in fruit treated

with 2 mM OA followed by 45 mM AA than other treatments. AA dipping caused decrease

in pH of the pericarp tissues of litchi fruit due to which PPO activity was retarded in treated

fruit as active sites of PPO was denatured at low pH (Tipton and Dixon, 1983). OA

application might have reduced the pH; therefore, PPO activity was low in OA-treated fruit

as activity of PPO enzyme is triggered by moisture loss and change in pH from acidic to

basic. Similarly, the peak activity of PPO enzyme was observed at 6.8 pH while at 4.0 pH its

activity was not detectable in ‘Huaizhi’ litchi (Jiang, 2000).


(Jiang and Fu, 1998). In current study, SSC of the litchi pulp tissues was increased with the

progression of cold storage period, irrespective to treatments. However, 2 mM OA and 45

mM AA application resulted in less increase in SSC than control fruit and 2 mM OA + 45

mM AA + 250 µL L-1 hexanal. OA and AA-treated fruit might have developed resistance to

moisture loss by improving membrane integrity and that’s why SSC of the fruit remained

stable. Similar results were also observed in ‘Bengal’, ‘Bedana’ and ‘Shahi’ litchi in which

SSC was increased with progression of storage period (Hojo et al., 2011; Aklimuzzaman et

al., 2011; Rajak et al., 2014). Concentration factor due to water loss or conversion of organic

acids to glycolytic intermediates and subsequently to sugars could be the reasons for increase

in SSC in treated fruit (Echeverria and Valich, 1989). Similar observation in SSC rate was

also reported by Tanada-Palmu and Grosso (2005), they pointed moisture loss as major

reason of increasing SSC as soluble contents were concentrated due to loss of water during

storage. Continuous decline in the TA of ‘Bedana’ litchi pulp tissues was observed

throughout storage period. However, 2 mM OA-treated fruit maintained significantly higher

TA, as compared to all other treatments. Previously, similar trend for TA was observed in

OA-treated mango fruit, where OA application maintained higher TA in contrast to untreated

fruit (Zheng et al., 2007a). Conversely, Hojo et al. (2011) reported that decline in

hydrochloric acid-treated litchi fruit could be due to consumption of organic acids in

respiration process.


152

TP and total antioxidants in litchi peel and pulp tissues were continuously decreased

throughout the cold storage period, but 2 mM OA-treated fruit maintained significantly

higher levels of TP and total antioxidants followed by 45 mM AA treatment, as compared to

other treatments. Higher PPO enzyme activity has been reported as major cause of decline in

phenolic compounds (Baltacig et al., 2011). Fruit treated with 2 mM OA and 45 mM AA

maintained higher TP in peel and pulp tissues than other treatments, by reducing the rate of

oxidation of phenolic compounds by reduced activities of PPO and POD enzymes.

Previously, AA treatment maintained higher level of TP by inhibiting PPO enzyme activities

and converting o-quinones back to phenolic substrates in fresh cut cantaloupe melon

(Lamikanra and Watson, 2001). Similarly, postharvest OA application has been reported to

increase antioxidant potential of pomegranate stored at 2°C for 84 days (Sayyari et al., 2010).

However, reduction in total antioxidants in litchi peel and pulp tissues with increase in

storage period could be due to oxygen stimulated oxidation of phenolic compounds (Stewart

et al., 1999). As total antioxidants are consumed in scavenging ROS species, under storage

conditions (Hounsome et al., 2009). Similarly, continuous reduction in ascorbic acid of litchi

pulp tissues was observed with increased storage period. Decreasing trend of AA contents

was also observed by (Hojo et al., 2011; Ray et al., 2005) also observed same behaviour of

AA contents of litchi fruit. However, 2 mM OA-treated maintained significantly higher AA

contents than all other treatments. Previously, acidic treatment maintained higher AA

contents in ‘Bengal’ litchi fruit (Silva et al., 2010).

SOD and CAT enzymes play important role in protecting antioxidants against ROS

(Niranjana et al., 2009), but their activities were decreased in litchi peel and pulp tissues, as

cold storage period progressed. However, 2 mM OA-treated fruit exhibited significant higher

activities of these enzymes, as compared to all other treatments. Higher activities of CAT and

SOD enzymes in OA-treated fruit resulted in increased antioxidative capacity of fruit against

oxidative stress, thereby, delayed senescence and pericarp browning of litchi fruit through

inhibition of lipid peroxidation (Zheng and Tian, 2006). Similarly, OA-treated peach fruit

exhibited significantly higher activities of SOD and CAT enzymes under ambient storage

conditions (Zheng et al., 2007b). On the other hand, activities of POD enzyme in peel and

pulp tissues was significantly increased with the enhancement of cold storage period.


153

However, 2 mM OA-treated fruit maintained significantly low activities of POD enzymes

both in peel and pulp tissues. Previously, Zheng and Tian (2006) observed lower POD

enzyme activities in OA-treated litchi fruit, as compared to untreated fruit.

9.6 Conclusion

Combination of 250 µL L-1 hexanal with 2 mM OA and 45 mM AA could not control

pericarp browning and quality of litchi cv. ‘Bedana’ fruit. However, exogenous application

of 2 mM OA delayed pericarp browning for longer period of time than any other treatment

along with better fruit quality and higher activities of antioxidative enzymes (SOD and CAT)

in litchi cv. ‘Bedana’ fruit during cold storage than other treatments.

General conclusions

154

General Conclusions

Litchi a non-climacteric fruit is getting popular among consumers and emerging as an

important crop for Pakistan. However, rapid pericarp browning causes reduction in its

cosmetic look within three days after harvest with reduced shelf and storage life. Therefore,

integrated research plan was made to investigate the effect of locations, cultivars, exogenous

application of chemicals like oxalic acid, ascorbic acid, hexanal and combination of the best

selected doses of all these chemicals on pericarp browning and fruit quality of litchi under

prolonged cold storage conditions. All the experiments were laid out into factorial

arrangements. To study the influence of harvest locations and cultivars on postharvest quality

of litchi, fruit were kept at ambient condition for 5 days after harvest. In independent

experiment, the effects of postharvest application of chemicals like oxalic acid, ascorbic acid

and hexanal on quality of litchi fruit were investigated during 28 days of cold storage. During

various experiments data were collected for fruit weight loss (from whole fruit); SSC, TA

and SS: TA ratio (from pulp tissues); pericarp browning index, activities of PPO enzymes

and anthocyanin contents (from peel tissues); whereas, TP, total antioxidants, activities of

POD, CAT and SOD enzymes (from both pulp and peel tissues) at alternate days during

study at ambient conditions and 7 days interval during cold storage. In first experiment, fruits

of litchi cv. ‘Gola’ and ‘Serai’ were harvested from two different locations (Lahore and

Haripur) to screen out best location for litchi fruit production and cultivar with less pericarp

browning and better fruit quality. Fruit were kept at ambient conditions for 5 days to evaluate

different physical, chemical and enzymatic changes. It was observed that fruit of litchi cv.

‘Gola’ exhibited less pericarp browning with superior fruit quality than ‘Serai’; while, fruit

harvested from Haripur location showed less pericarp browning with better fruit quality than

Lahore. On the basis of these results litchi cv. ‘Gola’ fruit harvested from Haripur location

was chosen for further experiments in which different chemicals were tested for their

influence on pericarp browning and activities of antioxidative enzymes. For this purpose

chemicals at different concentrations were selected based on their anti-browning properties

(oxalic acid and ascorbic acid) or abilities to improve shelf life (hexanal) were applied

exogenously in separate experiments to litchi fruit before storage. During confirmatory trials,

General conclusions

155

best concentration of all three chemicals and their combination were applied exogenously on

litchi cvs. ‘Gola’ and ‘Bedana’ fruits. Therefore, in second experiment litchi cv. ‘Gola’ fruit

harvested from Haripur location was subjected to postharvest oxalic acid treatments (0, 1, 2,

3 or 4 mM) to investigate the effect of oxalic acid on pericarp browning and quality of litchi

fruit. Among all the treatments, exogenous application of 2 mM oxalic acid significantly

delayed pericarp browning, improved physico-chemical attributes and maintained higher

activities of antioxidative enzymes (SOD and CAT) in litchi cv. ‘Gola’ fruit during 28 days

of cold storage. In third experiment, effect of different concentrations of ascorbic acid (0, 15,

30, 45 or 60 mM) on pericarp browning and fruit quality of litchi cv. ‘Gola’ fruit was

evaluated. Application of 45 mM ascorbic acid improved fruit quality by delaying pericarp

browning and maintaining better physico-chemical attributes along with higher activities of

antioxidative enzymes (SOD and CAT) in litchi cv. ‘Gola’ fruit during cold storage. In fourth

experiment influence of exogenous hexanal application (0, 250, 500, 750 or 1000 µL L-1) on

the pericarp browning, fruit quality and storage life of litchi cv. ‘Gola’ fruit was investigated.

Hexanal fumigated-fruit turned brown earlier than untreated fruit during cold storage period.

However, among different hexanal concentrations, 250 µL L-1 showed comparatively better

fruit quality attributes with higher activities of antioxidative enzymes (CAT and SOD) and

lower activities of PPO and POD enzymes, as compared to other hexanal treatments. From

above experiments, best concentrations of different chemicals (2 mM oxalic acid, 45 mM

ascorbic acid and 250 µL L-1 hexanal) were screened out and further used individually and in

combination (2 mM oxalic acid + 45 mM ascorbic acid + 250 µL L-1 hexanal) on another

litchi cv. ‘Bedana’ along with ‘Gola’ in separate experiments. Evidently, 2 mM oxalic acid

was remained more effective in delaying pericarp browning, improving fruit quality with

enhanced storage life of litchi cvs. ‘Gola’ and ‘Bedana’ fruits than all other treatments.

Combination of 250 µL L-1 hexanal with 2 mM oxalic acid and 45 mM ascorbic could not

control browning during cold storage. In conclusion, postharvest application of 2 mM oxalic

acid or 45 mM ascorbic acid was most effective chemical concentrations in delaying pericarp

browning and improving fruit quality under cold storage conditions on ‘Gola’ as well as

‘Bedana’ cvs. of litchi fruit.

General conclusions

156

Recommendations

The experimental data suggest that the postharvest practices can potentially delay

pericarp browning and improve fruit quality under cold storage conditions. Therefore, the

following recommendations are made to the litchi industry:

Fruit harvested from Haripur showed delayed browning and maintained better quality

than fruit harvested from Lahore. Therefore, litchi produced at Haripur location has

potential to have longer postharvest storage life with better fruit quality through

different postharvest management practices.

Dipping of litchi fruit in 2 mM oxalic acid or 45 mM ascorbic acid and then storage at

5oC with 90±5% RH should be considered safe for long term storage. However, with

increase storage period beyond three weeks, the risk of pericarp browning and quality

deterioration may increase.

Hexanal application alone or in combination with other anti-browning agents should

not be practiced as it desiccated litchi peel and fruit rapidly turned brown.

Disclaimer

These recommendations are purely based on the laboratory experiments conducted

under strictly controlled conditions. Discrepancies may arise in the outcomes under

commercial situations. The project investigator, Muhammad Shafique, and University of

Agriculture, Faisalabad, accept no liability whatsoever reason of negligence or otherwise

arising from the reliance or use of these recommendations.

Future Research

The effects of pre-harvest orchard management practices such as mineral nutrition

and irrigation need to be investigated for their potential roles in affecting the

postharvest pericarp browning and quality of litchi fruit.

General conclusions

157

A comprehensive study should also be conducted to evaluate the influence of the

investigated treatments on other commercial cultivars (‘Chinese’, ‘Litchi Siah’,

‘Madrasi’ and ‘Calcutti’) and harvest locations (Multan, Rahim Yar Khan, Khan Pur,

Moro, Mir Pur Khas and Tando Allah Yar) on postharvest pericarp browning and

quality of litchi fruit under extended cold storage period.

Research should be conducted to optimize post-storage temperature as litchi fruit

quality deteriorates rapidly after removing from cold storage.

There is need to investigate the relationship of postharvest pericarp browning in litchi

fruit with various enzymatic activities at molecular level to get better understanding

of the underlying mechanisms and their management.

References

158

REFERENCES

Ainsworth, E.A. and K.M. Gillespie. 2007. Estimation of total phenolic content and other

oxidation substrates in plant tissues using Folin-ciocalteu reagent. Nat. Protoc. 2:875-

877.

Akamine, E.K. 1960. Preventing the darkening of fresh litchi prepared for export. Hawaii

Agri. Exp. Stat. Univ., Tech. Prog. Report. 127:1-17.

Akamine, E.K. and T. Goo. 1977. Effects of gamma irradiation on shelf life of fresh lychees

(Litchi chinensis Sonn.). Hawaii Agric. Exp. Sta. Res. Bull. 169: 20p.

Akhtar, I., N.A. Abbasi and A. Hussain, 2010. Effect of calcium chloride treatments on

quality characteristics of loquat fruit during storage. Pak. J. Bot. 42:181-188.

Aklimuzzaman, MD., C. Goswami, J. Howlader, H.A. Kader and K. Hassan. 2011.

Postharvest storage behavior of litchi. J. Hort. Forest. Biotechnol. 15:1-8.

Altunkaya, A. and V. Gokmen. 2008. Effect of various inhibitors on enzymatic browning,

antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa). Food

Chem. 107:1173-1179.

Arberg, B. 1981. Plant growth regulators. Monosubstituted benzoic acid. Swed. Agric. Res.

11:93-105.

Baltacig, C., S. Veliog and E. Karacabey. 2011. Changes in total phenolic and flavonoid

contents of rowanberry fruit during postharvest storage. J. Food Quality. 34:278-283.

Barman, K., M.W. Siddiqui, V.B. Patel and M. Prasad. 2014. Nitric oxide reduces pericarp

browning and preserves bioactive antioxidants in litchi. Sci. Hortic. 171:71-77.

Batten, D.J. 1986. Harvesting litchis. p. 73-75. In: C.M. Menzel and G.N. Greer (ed.) The

Potential of Litchi in Australia. Proc. of the First National Litchi Seminar, 14-15

February 1986’ Sunshine Coast Tropical Fruits Association, Nambour Queensland,

Australia.

Batten, D.J. 1989. Maturity criteria for litchis (Lychees). J. Food Quality. 4:149-155.

References

159

Bhushan, B., A. Pal, R. Narwal, V.S. Meena, P.C. Sharma and J. Singh. 2015. Combinatorial

approaches for controlling pericarp browning in Litchi (Litchi chinensis) fruit. J. Food

Sci. Technol. 1-9.

Bloknina, O., E. Virolainen and K.V. Fagerstedt. 2003. Antioxidants, oxidative damage and

oxygen deprivation stress: A review. Annal. Bot. 91:179-194.

Brand-Williams, W., M.E. Cuvelier and C. Berset. 1995. Use of free radical method to

evaluate antioxidant activity. Lebensm Wiss Technol. 28:25-30.

Brouillard, R. 1982. Chemical structure of anthocyanins. p. 41-65. In: P. Markakis.

Anthocyanins as food colors. Academic Press, New York USA.

Brown, B.I. 1986. Postharvest handling and storage of lychees. p. 77-78. In: C.M. Menzel

and G.N. Greer (ed.) The Potential of Lychee in Australia. Proc. of the First National

Lychee Seminar, 14-15 February 1986. Sunshine Coast Tropical Fruits Association,

Nambour Queensland, Australia.

Bryant, P. 2004. Optimising the postharvest management of lychee (Litchi chinensis Sonn): a

study of mechanical injury and desiccation. Ph.D diss., Dept. Crop Sci., The Univ.

Sydnye, Australia.

Buta, J.G. and J. Abbott. 2000. Browning inhibition of three cultivars of fresh cut pears.

Hortic. Sci. 35:1111-1113.

Campbell, R.J. and C.W. Campbell. 2001. Longan evaluation and selection in Florida. Acta

Hortic. 558:125-127.

Cheema, A., P. Padmanabhan, J. Subramanian, T. Blom and G. Paliyath. 2014. Improving

quality of greenhouse tomato (Solanum lycopersicum L.) by pre- and postharvest

applications of hexanal-containing formulations. Postharvest Biol. Technol. 95:13-19.

Chen, F., Y.B. Li and M.D. Chen. 1986. The production of ethylene of litchi fruit during

storage and its control. Acta Hortic. Sin. 13:151-156.

Chen, H.J. and Q.Z. Hong. 1992. A study on the senescence and browning in the pericarp

of litchi (Litchi chinensis Sonn.) during storage. Acta Hortic. Sin. 19:227-232.

Chen, W. Wu., Z. Ji and M. Su. 2001. Postharvest research and handling of litchi in

China. Acta Hortic. 9:558-564.

References

160

Chen, Y.Z., P. Li, Y.R. Wang and L.P. Zeng. 1987. The effect of chilling temperature on

respiration, electrolyte leakage and cold storage life in litchi fruits. Acta Hortic. Sin.

14:169-173.

Delabarre, Y. 1989. Synthese bibliographique sur le ramboutan ou litchi chevelu (Nephelium

lappaceum L.) Fruits. 44:33-44.

Del-Aguila, J.S., P. Hofman, T. Campbell, J.R. Marques, L.S.H. Del Aguila and R.A. Kluge.

2009. Hydrocooling of 'B3' lychee fruit maintained in cold storage. Cienc. Rural.

39:273-2379.

De-Reuck, K., D. Sivakumar and L. Korsten. 2009. Integrated application of 1-

methylcyclopropene and modified atmosphere packaging to improve quality retention

of litchi cultivars during storage. Postharvest Biol. Technol. 52:71-77.

Ding, C.K., K. Chachin, Y. Ueda and C.Y. Wang. 2002. Inhibition of loquat enzymatic

browning by sulfhydryl compounds. Food Chem. 76:213-218.

Dong, Z.Y., J.W. Chi, G.M. Yang and X.J. Tang. 2005. The health effect and exploitation

prospect of litchi. Food Res. Develop. 5:148-151.

Duan, X.W., X.G. Su, Y.L. You, H.X. Qu, Y.B. Li, and Y.M. Jiang. 2007a. Effect of nitric

oxide on pericarp browning of harvested longan fruit in relation to phenolics

metabolism. Food Chem. 104:571-576.

Duan, X.W., Y.M. Jiang, X.G. Su, Z.Q. Zhang and J. Shi. 2007b. Antioxidant properties

of anthocyanins extracted from litchi (Litchi chinensis Sonn.) fruit pericarp tissues

in relation to their role in the pericarp browning. Food Chem. 101:1382-1388.

Duan, X.W., Y.M. Jiang, X.G. Su and Z.Q. Zhang. 2004. Effects of pure oxygen on

enzymatic browning and quality of postharvest litchi fruit. J. Hortic. Sci.

Biotechnol. 79:859-862.

Ducamp-Collin, M.N., H. Ramarson, M. Lebrun, G. Self and M. Reynes. 2008. Effect of

citric acid and chitosan on maintaining red colouration of litchi pericarp. Postharvest

Biol. Technol. 49:241-246.

Duthie, G. and A. Crozier. 2000. Plant derived phenolic antioxidants. Curr. Opin. Lipidol.

11:43-47.

References

161

EAFUS: A Food Additive Database.2006. Center of Food Safety and Applied Nutrition. U.S.

Food and Drug Administration (FDA).

Echeverria, E. and J. Valich. 1989. Enzymes of sugar and acid metabolism in stored'

Valencia' oranges. J. Am. Soc. Hortic. Sci. 12:745-758.

Fallik, E., S. Grinberg, S. Alkalai, O. Yekutieli, A. Wiseblum, R. Regev, H. Beres and E.

Bar-Lev. 1999. A unique rapid hot water treatment to improve storage quality of

sweet pepper. Postharvest Biol. Technol. 15:25-32.

Fitzell, R.D. and L.M. Coates. 1995. Diseases of lychee. p. 41-42. In: L. Coates, T. Cooke,

D. Persley, B. Beattie, N. Wade and R. Ridgeway (ed.). Postharvest diseases of

horticultural produce, tropical fruits, QLD Primary Industries, Brisbane, Australia.

Follett, P.A. and S.S. Sanxter. 2003. Lychee quality after hot-water immersion and X-ray

irradiation quarantine treatments. HortScience. 38:1162.

Gao, J.C, M.Y. Yuan and R.J. Xu. 1990. Postharvest physiology and anti-browning of litchi

fruit. Food Sci. 6:49-52.

Gimnez, M., C. Olarte, S. Sanz, C. Lomas, L. Echavarri and F. Ayala. 2003. Influence of

packaging films on the sensory and microbiological evolution of minimally processed

borage (Borrago officinalis). J. Food Sci. 68:1051-1058.

Gonzalez-Aguilar, G.A., S. Ruiz-Cruz, H. Soto-Valdez, F. Vazquez-Ortiz, R. Pacheco-

Aguilar and C.Y. Wang. 2005. Biochemical changes of fresh-cut pineapple slices

treated with antibrowning agents. Int. J. Food Sci. Technol. 40:377-383.

Guerra, M. and P.A. Casquero. 2009. Site and fruit maturity influence on the quality of

European plum in organic production. Sci. Hortic. 122:540-544.

Guerrero-Beltran, J.A., B.G. Swanson and G.V. Barbosa-Canovas. 2005. Inhibition of

polyphenol oxidase in mango puree with 4-hexylresorcinol, cysteine and ascorbic

acid. Food Sci. Technol. 38:625-630.

Hajare, S.N., S. Saxena, S. Kumar, S. Wadhawan, V. More and B.B. Mishra. 2010. Quality

profile of litchi (Litchi chinensis Sonn.) cultivars from India and effect of radiation

processing. Radiat. Phys. Chem. 79:994-1004.

Han, D.M., Z.X. Wu, Z.L. Ji and M. Han. 1999. Effects of SO2 treatment on the oxidation

and senescence of longan fruit. J. Fruit Sci. 16:24-29.

References

162

Hodges, D.M. 2003. Postharvest oxidative stress in horticultural crops. New York Food

Production Press. 284p.

Hojo, E.T.D., J.F. Durigan, R.H. Hojo, J.R. Donadon and R.N. Martins. 2011. Use of

hydrothermal and acid treatment in the quality of litchi ‘Bengal’. Rev. Bras. Frutic.

33:386-393.

Holcroft, D.M. and E.J. Mitcham. 1996. Postharvest physiology and handling of litchi (Litchi

chinensis Sonn.). Postharvest Biol. Technol. 9:265-281.

Hong, Q.Z., Z.J. Xie and W.J. Chen. 1986. Investigations on the preservation of

postharvest litchi (Litchi chinensis Sonn.) fruits. J. Fujian Agric. College 15:19-

27.

Hortwitz, W. 1960. Official and tentative methods of analysis. Association of the Official

Agriculture Chemist, Washington, D.C. Ed. 9:320-341.

Hounsome, N., B. Hounsome, D. Tomos and G. Edwards-Jones. 2009. Changes in

antioxidant compounds in white cabbage during winter storage. Postharvest Biol.

Technol. 52:173-179.

Hu, Z.Q., X.M. Huang, H.B. Chen and H.C. Wang. 2010. Antioxidant capacity and

phenolic compounds in litchi (Litchi chinensis sonn.) pericarp. Acta. Hortic. 863:567-

574.

Hu, W.R., X.Q. Pang, S.Z. Liu, Z.Q. Zhang and Z.L. Ji. 2005. Effect of postharvest handling

on the anthocyanin content and anthocyanase activity in litchi pericarp. J. Fruit Sci.

22:224-228.

Huang, P.Y. and K.J. Scott. 1985. Control of rotting and browning of litchi fruit after harvest

at ambient temperature in China. Trop. Agric. 62:2-4.

Huang, C., and Y. Wang. 1989. Effect of storage temperature on the color and quality of

litchi fruit. Acta Hortic. 269:307-308.

Huang, H., G. Jing, L. Guo, D. Zhang, B. Yang, X. Duan, M. Ashraf and Y. Jiang. 2013.

Effect of oxalic acid on ripening attributes of banana fruit during storage. Postharvest


Huang, S., H. Hart, H. Lee and L. Wicker. 1990. Enzymatic and colour changes during

postharvest storage of lychee fruit. J. Food Sci. 55:1762-1763.

References

163

Huang, X., H.C. Wang, J. Li, J. Yin, W. Yuan, J. Lu and H.B. Huang. 2003. An overview of

calcium’s role in litchi fruit cracking. Acta Hortic. 665:231-240.

Ilangantileke, S.G., A. Noomhorn, I.P. Upadhyay and M.S. Rao. 1993. Effect of irradiation

and storage temperature on the shelf life and quality of Thai lychee. p. 352-354. In:

B.R. Champ, E. Highley and G.I. Johnson (ed.) Postharvest handling of tropical

fruits. ACIAR Conference Proc. Chang Mai, Thailand.

Isabelle, M. B.L. Lee, M.T. Lim, W.P. Koh, D. Huang and C.N. Ong. 2010. Antioxidant

activity and profiles of common fruits in Singapore. Food Chem. 123:77-84.

Jacobi, K.K., L.S. Wong and J.E. Giles. 1993. Lychee (Litchi chinensis Sonn.) fruit quality

following vapour heat treatment and cool storage. Postharvest Biol. Technol. 3:111-

119.

Jaiswal, B.P., N.L. Shah and U.S. Prasad. 1987. Regulation of colour break during litchi

(Litchi chinensis Sonn.) ripening. Indian J. Exp. Biol. 25:66-72.

Ji, Z.L., L.F. Liang, J.L. Liu and G.T. Wang. 1992. Studies on the changes of endogenous

hormone contents in litchi (Litchi chinensis Sonn.) fruits during development. J.

South China Agric. Univ. 13:93-98.

Jiang, J.P., M.X. Su and P. Lee. 1986. The production and physiological effects of ethylene

during ontogeny and after harvest of litchi fruits. Acta Phytophysiol. Sinica. 12:95-

103.

Jiang, Y. 2000. Role of anthocyanin, polyphenol oxidase and phenols in lychee pericarp

browning. J. Sci. Food Agric. 80:305-310.

Jiang, Y., Y. Li and L. Jianrong. 2004. Browning control, shelf life extension and quality

maintenance of frozen litchi fruit by hydrochloric acid. J. Food Eng. 63:147-151.

Jiang, Y.M. and F. Chen. 1995. A study on polyamine change and browning of fruit during

cold storage of litchi (Litchi chinensis Sonn.). Postharvest Biol. Technol. 5:245-250.

Jiang, Y.M. and J.R. Fu. 1998. Inhibition of polyphenol oxidase and the browning control of

litchi fruit by glutathione and citric acid. Food Chem. 62:49-52.

Jiang, Y.M. and J.R. Fu. 1999. Postharvest browning of litchi fruit by water loss and its

prevention by controlled atmosphere storage at high relative humidity. LWT-Food

Sci. Technol. 32:278-283.

References

164

Jiang, Y.M. and Y.B. Li. 2003. Effect of low temperature acclimation on browning of

litchi fruit in relation to shelf life. J. Hortic. Sci. 78:437-440.

Jiang, Y.M., G. Zauberman and Y. Fuchs. 1997. Partial purification and some properties of

polyphenol oxidase extracted from litchi pericarp. Postharvest Biol. Technol. 10:221-

226.

Jiang, Y.M., J. Li and W. Jiang. 2005. Effects of chitosan coating on shelf life of cold stored

litchi fruit at ambient temperature. LWT-Food Sci. Technol. 38:757-761.

Jiang, Y.M., L.H. Yao, A. Lichter and J.R. Li. 2003. Postharvest biology and technology of

litchi fruit. J. Food Agric. Environ. 1:76-81.

Jiang, Y.M., Y. Wang, L. Song, H. Liu, A. Lichter, O. Kerdchoechuen, D.C. Joyce and J.

Shi. 2006. Postharvest characteristics and handling of litchi fruit - an overview. Aust.

J. Exp. Agric. 46:1541-1556.

Jiang, Y.M., Z.Q. Zhang, D.C.M. Joyce and S. Ketsa. 2002. Postharvest biology and

handling of longan fruit (Dimocarpus Longan Lour.). Postharvest Biol. Technol.

26:241-252.

Jimenez, A., J.A. Hernandez, L.A. Rioand and F. Sevilla. 1997. Evidence for the presence of

the ascorbate–glutathione-cycle in mitochondria and peroxisomes of pea leaves.

Plant. Physiol. 11:4275-284.

Joas, J., Y. Caro, M.N. Ducamp and M. Reynes. 2005. Postharvest control of pericarp

browning of litchi fruit (Litchi chinensis Sonn. cv. Kwai Mi) by treatment with

chitosan and organic acids. I. Effect of pH and pericarp dehydration. Postharvest Biol.

Technol. 38:128-136.

Joubert, A.J. 1970. The litchi. Bull. Dep. Agric. Technol. Serv. S. Afr. 389:1-22.

Joubert, A.J. 1986. Litchi. p. 233-246. In: S.P. Monselise (ed.). Fruit set and development.

CRC Press Inc., Boca Raton.

Joubert, A.J. and Van Lelyveld. 1975. An investigation of pre-harvest browning of litchi

peel. Phytophylactica. 7:9-14.

Joyce, D. and B. Patterson. 1994. Postharvest Water Relations in Horticultural Crops:

Principles and Problems: Australian Centre for International Agricultural Research,

Australia.

References

165

Jung, S.K. and C. Watkins. 2008. Superficial scald control after delayed treatment of apple

fruit with diphenylamine (DPA) and 1-methylcyclopropene (1-MCP). Postharvest


Kahn, V. 1985. Tropolone-a compound that can aid in differentiating between tyrosinase and

peroxidase. Phytochemistry. 24:915-920.

Kanazawa, S., S. Sano, T. Koshiba and T. Ushimaru. 2000. Changes in antioxidative

enzymes in cucumber cotyledons during natural senescence: comparison with those

during dark induced senescence. Physiol. Plantarum. 109:211-216.

Karlidag, H., E. Yildirim and M. Turan. 2009. Exogenous applications of salicylic acid affect

quality and yield of strawberry grown under antifrost heated greenhouse conditions.

J. Plant Nutr. Soil Sci.172:270-276.

Kayashima, T. and T. Katayama. 2002. Oxalic acid is available as a natural antioxidant in

some systems. Biochim. Biophys. Acta. 1573:1-3.

Khalid, S., A.U. Malik, A.S. Khan and A. Jamil. 2012. Influence of exogenous application of

plant growth regulators on fruit quality of young ‘Kinnow’ mandarin (Citrus nobilis x

C. deliciosa) tress. Int. J. Agric. Biol. 14:229-234.

Khan, A.S., N. Ahmad, A.U. Malik and M. Amjad. 2012. Cold storage influences the

postharvest pericarp browning and quality of litchi. Int. J. Agric. Biol. 14:389-394.

Kitts, D.D. 1997. An evaluation of the multiple effects of the antioxidant vitamins. Trends

Food Sci. Technol. 8:198-203.

Knee, M. and S.M. Smith. 1989. Variation in quality of apple fruits stored after harvest on

different dates. J. Hortic. Sci. 64:413-419.

Kramer, M.S. and G.D. Kuhn. 1964. The effect of radiation on mold populations on fresh

lychees. Proc. Fla. State Hortic. Soc. 77:436-438.

Ku, V.V.V., R.B.H. Wills and Y.Y. Leshem. 2000. Use of nitric oxide to reduce

postharvest water loss from horticultural produce. J. Hortic. Sci. Biotechnol.

75:268-270.

Kudachikar, V.B., M.T. Semeerbabu, B. Revathy, A. Ushadevi, K.V.R. Ramana and R.S.

Matches. 2007. Effect of postharvest treatments on shelf life and quality of litchi fruit

References

166

stored under modified atmosphere at low temperature. J. Food Sci. Technol. 44:106-

109.

Kumar, D., D.S. Mishra, B. Chakraborty and P. Kumar. 2013. Pericarp browning and quality

management of litchi fruit by antioxidants and salicylic acid during ambient storage.

J. Food Sci. Technol. 50:797-802.

Kumar, S., B.B. Mishra, S. Saxena, N. Bandyopadhyay, V. More, S. Wadhawan, S.N.

Hajare, S. Gautam and A. Sharma. 2012. Inhibition of pericarp browning and shelf

life extension of litchi by combination dip treatment and radiation processing. Food

Chem. 131:1223-1232.

Kumcha, U. 1998. The effects of cultural practices and environmental factors on fruit

development in litchi (Litchi chinensis Sonn.). Ph. D diss., University of Queensland,

Australia.

Lam, P.F., S. Kosiyachinda, M.C.C. Lixada, D.B. Mendoza, S. Prabawati and S.K. Lee.

1987. Postharvest physiology and storage of rambutan. p. 39-50. In: P.F. Lam and S.

Kosiyachinda (ed.). Rambutan fruit development, postharvest physiology and

marketing in ASEAN. Kuala Lumpur, Malaysia.

Lamattina, L., C. García-Mata, M. Graziano and G. Pagnussat. 2003. Nitric oxide: the

versatility of an extensive signal molecule. Annu. Rev. Plant Biol. 54:109-136.

Lamikanra, O. and M.A. Watson. 2001. Effects of ascorbic acid on peroxidase and

polyphenoloxidase activities in fresh‐cut cantaloupe melon. J. Food Sci. 66:1283-

1286.

Lanciotti, R., M.R. Corbo, F. Gardini, M. Sinigaglia and M.E. Guerzoni. 1999. Effect of

hexanal on the shelf life of fresh apple slices. J. Agric. Food Chem. 47:4769-4776.

Landrigan, M., S.C. Morris, D. Eamus and W.B. McGlasson. 1996. Postharvest water

relationship and tissue browning of rambutan fruits. Sci. Hortic. 66:201-208.

Landrigan, M., V. Sarafis, S.C. Morris and W.B. mcGlasson. 1994. Structural aspects of

Rambutan (Nephelium lappaceum L.) fruits and their relation to postharvest

browning. J. Hortic. Sci. 69:571-579.

Lee, H.S. and L. Wicker. 1991. Quantitative changes in anthocyanin pigments of lychee fruit

during refrigerated storage. Food Chem. 40:263-270.

References

167

Lee, S.K. and A.A. Kader. 2000. Pre-harvest and postharvest factors influencing vitamin C

content of horticultural crops. Postharvest Biol. Technol. 20:207-220.

Lei, D.F., F. Feng and D.Z. Jiang. 2004. Characterization of polyphenol oxidase from plants.

Prog. Nat. Sci. 14:553-561.

Liang, H.H., Z.L. Ji and X.Y. Huang. 1998. Study on the techniques of package and

storage for litchi fruit stored in the room temperature. J. Fruit Sci. 15:158-163.

Lichter, A., O. Dvir, I. Rot, M. Akerman, R. Regev, A. Wiesblum, E. Fallik, G. Zauberman

and Y. Fuchs. 2000. Hot water brushing: an alternative method to SO2 fumigation for

color retention of litchi fruits. Postharvest Biol. Technol. 18:235-244.

Lima, R.A.Z., C.M. Abreu, S.A. Asmar, A.D. Corrêa and C.D. Santos. 2010. Packing and

covering in lychee (Litchi chinensis Sonn.) stored under uncontrolled conditions.

Ciênc. Agrotec. 34:914-921.

Lin, H.T. 2002. Postharvest pericarp browning mechanism and postharvest handling of

Longan fruit. Ph. D diss., Zhejiang Univ. Hangzhou, China.

Lin, H.T., Y.F. Xi and S.J. Chen. 2005. The relationship between the desiccation-induced

browning and the metabolism of active oxygen and phenolics in pericarp of

postharvest longan fruit. J. Plant Physiol. Mol. Biol. 31:287-297.

Lin, Z.F., S.S. Li, D.L. Zhang, S.X. Liu, Y.B. Li, G.Z. Lin and M.D. Chen. 1988. The

changes of oxidation and peroxidation in postharvest litchi fruit. Acta Bot. Sin.

30:383-387.

Liu, D., J. Zou, Q. Meng, J. Zou and W. Jiang. 2009. Uptake and accumulation and oxidative

stress in garlic (Allium sativum L.) under lead phytotoxicity. Ecotoxicology. 18:134-

143.

Liu, H., J. Shi, L. Song, Y. You and Y. Jiang. 2006. Browning control and quality

maintenance of litchi fruit treated with combination of N-acetyl cysteine and

isoascorbic acid. J. Food Technol. 4:147-151.

Luton, M.T. and D.A. Holland. 1986. The effects of preharvest factors on the quality of

stored pears. J. Hortic. Sci. 61:23-32.

Mahajan, B.V.C. 1997. Studies on the bio-chemical changes in litchi fruits during storage.

Ind. J. Plant Physiol. 2:310-311.

References

168

Mahajan, P.V. and T.K. Goswami. 2004. Extended storage life of litchi fruit using controlled

atmosphere and low temperature. J. Food Process. Pres. 28:388-403.

Mangaraj, S., T.K. Goswami, S.K. Giri, and M.K. Tripathi. 2012. Permselective MA

packaging of litchi (cv. Shahi) for preserving quality and extension of shelf-life.

Postharvest Biol. Technol. 71:1-12.

Manjunatha, G., V. Lokesh and B. Neelwarne. 2010. Nitric oxide in fruit ripening: trends

and opportunities. Biotechnol. Adv. 28:489-499.

Marangoni A.G., T. Palma and D.W. Sanley. 1996. Membrane effects in postharvest

physiology. Postharvest Biol. Technol. 7:193-217.

Markakis, T. and F. Skoog. 1982. Anthocyanin as food colours. New York: Academic Press.

Martínez-Castellanos, G., K. Shirai, C. Pelayo-Zaldívar, L.J. Pérez-Flores and J.D.

Sepúlveda-Sánchez. 2009. Effect of Lactobacillus plantarum and chitosan in the

reduction of browning of pericarp Rambutan. Food Microbiol. 26:444-449.

Mathew, A.G. and H.A.B. Parpia. 1971. Food browning as a polyphenol reaction. Adv. Food

Res. 19:75-145.

Mathew, M.D., D. Kleinhenz and J.G. Streeter. 2005. Temperature and cultivar effects on

anthocyanin and chlorophyll b concentrations in three related Lollo lettuce cultivars.

HortScience. 40:1731-1733.

Mayer, A.M. and E. Harel. 1979. Polyphenol oxidase in plants. Phytochemistry. 18:193-215.

McLauchlan, R.L., G.E. Mitchell, G.I. Johnson, S.M. Nottingham and K.M. Hammerton.

1992. Effects of disinfestation-dose irradiation on the physiology of Tai So lychee.


Menzel, C. 2001. The physiology of growth and cropping in lychee. South African Litchi

Growers’ Association Yearbook 12:9-14.

Mitra, S. and N. Kar. 2001. Organoleptic rating and physiological loss in weight during

storage of litchi fruit cv. ‘Bombai’. Environ. Ecol. 19:239-240.

Mitra, S., A.B.S. Harangi and N. Kar. 1996. Effect of polyethylene at low temperature

and different growth regulators at ambient temperature on changes in total soluble

solids, total sugar, titratable acidity and ascorbic acid content of litchi (cv.

Bombai) during storage. Environ. Ecol. 14:538-542.

References

169

Mizobutsi, G.P., F.L. Finger, R.A. Ribeiro, R. Puschmann, L.L.M. Neves and W.F. Mota.

2010. Effect of pH and temperature on peroxidase and polyphenoloxidase activities of

litchi pericarp. Sci. Agric. 67:213-217.

Monsalve-Gonzalez, A., Barbosa-Canovas, A. Mcevily and R. Lyengar. 1995. Inhibition of

enzymatic browning in apple products by 4-hexylresorcinol. Food Technol. 49:110-

118.

Mozafar, A. 1994. Plant Vitamins: Agronomic, Physiological and Nutritional Aspects. CRC

Press, Boca Raton, London.

Nagar, R.K. 1994. Physiological and biochemical studies during fruit ripening in litchi

(Litchi chinensis Sonn.). Postharvest Biol. Technol. 4:225-234.

Narayana, C.K., R.K. Pal and S.K. Roy. 1996. Effect of pre-storage treatments and

temperature regimes on shelf-life and respiratory behaviour of ripe Baneshan mango.

J. Food Sci. Technol. 33:79-82.

Neil, S.J., R. Desikan and J.T. Hancock. 2003. Nitric oxide signaling in plants. New Phytol.

159:11-35.

Neri, F., M. Mari and S. Brigati. 2006. Control of Penicillium expansum by plant volatile

compounds. Plant Pathol. 55:100-105.

Nicholls, B.G. 2001. A perspective of the Australian longan industry. Acta Hortic. 558:45-

47.

Nip, W. 1988. Handing and preservation of litchi (Litchi chinensis Sonn.) with emphasis on

colour. Trop. Sci. 28:5-11.

Niranjana, P., R.K.P. Gopalakrishna, R.D.V. Sudhakar and B. Madhusudhan, 2009. Effect of

controlled atmosphere storage (CAS) on anioxidant enzymes and DPPH-Radical

scavenging activity of mango (Mangifera indica L.) cv. Alphonso. Afr. J. Food Agri.

Nutr. Develop. 9:779-792.

Olesen, T. and N. Wiltshire. 2000. Postharvest results from the 1999/2000 litchi season.

Living Lychee. 23:16-22.

Olesen, T., L. Nacey, N. Wiltshire, and S. O’Brien. 2004. Hot water treatments for the

control of rots on harvested litchi (Litchi chinensis Sonn.) fruit. Postharvest Biol.

Technol. 32:135-146.

References

170

Paliyath, G. and J. Subramanian. 2008. Phospholipase D inhibition technology for enhancing

shelf life and quality. p. 195-239. In: G. Paliyath, D.P. Murr, A.K. Handa and S. Lurie

(ed.). Postharvest biology and technology of fruits, vegetable, and flowers. Wiley-

Blackwell, USA.

Pang, X.Q., X.W. Duan, Z.Q. Zhang, F.C Xu and Z.L. Ji. 2004. Purification and some

properties of peroxidase from pericarp of litchi (Litchi chinensis Sonn.). J. Trop.

Subtrop. Bot. 12:449-454.

Pantastico, E.B., J.B. Pantastico and V.B. Cosico. 1975. Some forms and functions of the

fruit and vegetable epidermis. Kalikasan Philipp. J. Biol. 4:175-197.

Partridge, I. 1997. Tropical fruit becomes major industry. Rural Res. 74:9-12.

Paull, R.E. and N.J. Chen. 1987. Changes in longan and rambutan during postharvest storage.

HortScience. 22:1303-1304.

Paull, R.E., M.E.Q. Reyes and M.U. Reyes. 1995. Litchi and rambutan insect disinfestation

treatments to minimize induced pericarp browning. Postharvest Biol. Technol. 6:139-

148.

Peel, M.C., B.L. Finlayson and T.A. McMahon. 2007. Updated world map of the

KöppenGeiger climate classification. Hydrol. Earth Syst. Sci. Discuss. 4:439-473.

Peng, L. and Y. Jiang. 2006. Exogenous salicylic acid inhibits browning of fresh-cut Chinese

water chestnut. Food Chem. 94:535-540.

Peng, Y.H. and W. Cheng. 1999. Review on fruit postharvest technology. J. Fruit Sci.

16:293-300.

Popova, L., T. Pancheva and A. Uzunova. 1997. Salicylic acid: Properties, biosynthesis and

physiological role. Bulg. J. Plant Physiol. 23:85-93.

Pornchaloempong, P., S.A. Sargen and A.J. Fox. 1998. Controlled atmosphere storage

can extend postharvest quality of lychee fruit (cv. ‘Mauritius’). Proc. Fla. State.

Hortic. Soc. 111:238-243.

Pornchaloempong, P., S.A. Sargent and C.L. Moretti. 1997. Cooling method and shipping

container affect lychee fruit quality. Proc. Fla. State Hortic. Soc. 110:197-200.

References

171

Prasad, N.K., B. Yang, M. Zhao, B.S. Wang, F. Chen and Y. Jiang. 2009. Effects of

high‐pressure treatment on the extraction yield, phenolic content and antioxidant

activity of litchi (Litchi chinensis Sonn.) fruit pericarp. Int. J. Food Sci.

Technol. 44:960-966.

Proctor, J.T.A. 1974. Colour stimulation in attached apples with supplementary light. J. Plant

Sci. 54:499-503.

Prusky, D., Y. Fuchs, I. Kobiler, I. Roth, A. Weksler, Y. Shalom, E. Fallik, G. Zauberman, E.

Pesis, and M. Akerman. 1999. Effect of hot water brushing, prochloraz treatment and

waxing on the incidence of black spot decay caused by Alternaria alternate in mango

fruit. Postharvest. Biol. Technol. 15:165-174.

Rajak, D., P.D. Sharma and K. Sanjeev. 2014. Effect of total soluble solid during storage of

litchi fruits under different temperatures. Adv. Appl. Sci. Res. 5:117-121.

Rajwana, I.A., A.U. Malik, A.S. Khan and R. Anwar. 2010. Lychee industry in Pakistan:

potential and prospects. Acta Hortic. 863:67-72.

Rao, M.V., G. Paliyath and D.P. Ormrod. 1996. Ultraviolet-B-and ozone-induced

biochemical changes in antioxidant enzymes of Arabidopsis thaliana. Plant Physiol.

110:125-136.

Raskin, I. 1992. Role of salicylic acid in plants. Ann. Rev. Plant. Physiol. Mol. Biol. 43:439-

463.

Ray, P.K. 1998. Postharvest handling of litchi fruits in relation to colour retention. A critical

appraisal. J. Food. Sci. Technol. 35:103-116.

Ray, P.K., R. Ruby and S.K. Singh. 2005. Effect of sulphur dioxide fumigation and low

temperature storage on post-harvest browning and quality of litchi fruits. Food Sci.

Technol. 42:226-230.

Razzaq, K., A.S. Khan, A.U. Malik and M. Shahid. 2013. Ripening period influences fruit

softening and antioxidative system of ‘Samar Bahisht Chaunsa’ mango. Sci. Hortic.

160:108-114.

Reshi, M., R.K. Kaul, A. Bhat and S.K. Sharma. 2014. Response of postharvest treatments

on nutritional characteristics and shelf life of litchi (cv. Dehradun). The Bioscan.

8:1219-1222.

References

172

Robards, K., P.D. Prenzler, G. Tucker, P. Swatsitang and W. Glover. 1999. Phenolic

compounds and their role in oxidative processes in fruits. Food Chem. 66:401-436.

Robert, C., F. Soliva and O.B. Martın. 2003. New advances in extending the shelf life of

fresh-cut fruits: Trends Food Sci. Tech. 14:341-353.

Rojas-Grau, M.A., A. Sobrino-Lopez, M.S. Tapia and O. Martin-Belloso. 2006. Browning

inhibition in fresh-cut ‘Fuji’ apple slices by natural antibrowning agents. J. Food Sci.

71:59-65.

Rub, A., S. Haq, S.A. Khalil and S.G. Ali. 2010. Fruit quality and senescence related changes

in sweet orange cultivar Blood red unipacked in different packing materials. Sarhad J.

Agric. 26:221-227.

Ruenroengklin, N., J. Zhong, X. Duan, B. Yang, J. Li and Y. Jiang. 2008. Effects of various

temperatures and pH values on the extraction yield of phenolics from litchi fruit

pericarp tissue and the antioxidant activity of the extracted anthocyanins. Int. J. Mol.

Sci. 9:1333-1341.

Saengnil, K., K. Lueangprasert and J. Uthaibutra. 2006. Control of enzymatic browning of

harvested ‘Hong Huay’ litchi fruit with hot water and oxalic acid dips. ScienceAsia.

32:345-350.

Sams, C.E. 1999. Preharvest factors affecting postharvest texture. Postharvest Biol. Technol.

15:249-254.

Santerre, C.R., J.N. Cash and D.J. Vannorman. 1988. Ascorbic acid combinations in the

processing of frozen apple slices. J. Food Sci. 53:1713-1716.

Sapers, G.M., K.B. Hicks, J.G. Phillips, L. Garzarella, D.L. Pondish T.J. McCormick S.M.

Sondey, P.A. Sci and Y.S. El-Ataway. 1989. Control of enzymatic browning in apple

with ascorbic acid derivatives, polyphenol oxidase inhibitors and complexing agents.

J. Food Sci. 54:997-1002.

Sayyari, M., D. Valero, M. Babalar, S. Kalantari, P.J. Zapata and M. Serrano. 2010. Pre-

storage oxalic acid treatment maintained visual quality, bioactive compounds, and

antioxidant potential of pomegranate after long-term storage at 2°C. J. Agric. Food

Chem. 58:6804-6808.

References

173

Scott, K.J., B.I. Brown, G.R. Chaplin, M.E. Wilcox and J.M. Bain. 1982. The control of

rotting and browning of litchi fruit by hot benomyl and plastic film. Sci. Hortic.

16:253-262.

Seifried, H.E., D.E. Anderson, E.I. Fisher and J.A. Milner. 2007. A review of the interaction

among dietary antioxidants and reactive oxygen species. J. Nutr. Biochem 18:567-

579.

Shah, N.S. and N. Nath, 2008. Changes in qualities of minimally processed litchis: Effect of

antibrowning agents, osmo-vacuum drying and moderate vacuum packaging. Dept.

Food Sci. Technol., G.B. Pant Uni. Agric. Technol. Pantnagar 263145, India.

Shah, S.S.H. 2003. Fruit orchard survey in district Haripur. Project for Horticulture

Promotion (East), Abbottabad, NWFP, Pakistan. p.14-15.

Shewfelt, R.L. 1990. Sources of variation in the nutrient content of agricultural commodities

from the farm to the consumer. J. Food Quality. 13:37-54.

Shewfelt, R.L. 1993. Measuring quality and maturity. p. 99-123. In: R.L. Shewfelt and S.E.

Prussia (ed.). Postharvest handling: A systems approach, Academic Press, Inc.,

California, USA.

Shi, J.X., C.S. Wang, X.Z. An, J.H. Li and M. Zhao. 2001. Postharvest Physiology, Storage

and Transportation of Litchi Fruit-A Review. Acta Hortic. p. 387-392.

Shiri, M.L., M. Ghasemnezhad, D. Bakhshi and M. Dadi. 2011. Changes in phenolic

compounds and antioxidant capacity of fresh-cut table grape (Vitis vinifera) cultivar

‘Shahaneh’ as influence by fruit preparation methods and packagings. Aust. J. Crop

Sci. 12:1515-1520.

Shirra, M., G. D’hallewin, S. Ben-Yehoshua and E. Fallik. 2000. Host-pathogen interactions

modulated by heat treatment. Postharvest Biol. Technol. 21:71-85.

Sholberg, P. and P. Randall. 2005. Hexanal vapor for postharvest decay control and aroma

production in stored pome fruit. Phytopathol. 4:95-96.

Shuecheng, L. 1981. Studies on transit and storage methods of litchi. J. Agric. Res. China.

30:251-260.

References

174

Silva, D.F.P.D., L.C.R.D. Lins, E.C. Cabrini, B.G. Brasileiro and L.C.C. Solomon. 2012.

Influence of the use of acids and films in post-harvest lychee conservation. Food Sci.

Technol. 59:745-750.

Silva, D.F.P.D., E.C.A. Cabrini, R.R. Alves and L.C.C. Salomao. 2010. Use of ascorbic acid

in the control of browning in lychees pericarp. Rev. Bras. Frutic. 32:618-627.

Simic, M.G. 1988. Mechanisms of inhibition of free-radical processes in mutagenesis and

carcinogenesis. Mutat. Res-Fund. Mol. M. 202:377-386.

Sivakumar, D., C.A. Terry and L. Korsten. 2010. An overview on litchi fruit quality and

alternative postharvest treatments to replace sulphur dioxide fumigation. Food

Rev. Int. 26:162-188.

Sivakumar, D. and L. Korsten. 2010. Fruit quality and physiological responses of litchi

cultivar McLean’s Red to 1-methylcyclopropene pre-treatment and controlled

atmosphere storage conditions. LWT-Food Sci. Technol. 43:942-948.

Sivakumar, D., E. Arrebola and L. Korsten. 2008. Postharvest decay control and quality

retention in litchi (cv. McLean's Red) by combined application of modified

atmosphere packaging and antimicrobial agents. Crop Prot. 27:1208-1214.

Sivakumar, D., L. Korsten and K. Zeeman. 2007. Postharvest management on quality

retention of litchi during storage. Fresh Produce. 1:66-75.

Sivakumar, D. and L. Korsten. 2006. Evaluation of the integrated application of two types of

modified atmosphere packaging and hot water treatments on quality retention in the

litchi cultivar McLean's Red. J. Hortic. Sci. Biotechnol. 81:639-644.

Sivakumar, D., T. Regnier, B. Demoz and L. Korsten. 2005. Effect of different postharvest

treatments on overall quality retention in litchi fruit during low temperature storage. J.

Hortic. Sci. Biotech. 80:32-38.

Snowdon, A.L. 1990. A colour atlas of post-harvest diseases and disorders of fruit and

vegetables: General introduction and fruits. 1st ed. Wolfe Scientific, Barcelona Spain.

Salomao, L.C.C., D.L.D. Siqueira, M.E.C. Pereira and P.R.G. Pereira. 2006. Macro and

micronutrients accumulation in inflorescences and fruits of the litchi ‘Bengal’. Cienc.

Rural. 36:793-800.

References

175

Somboonkaew, N. and L.A. Terry. 2010. Physiological and biochemical profiles of imported

litchi fruit under modified atmosphere packaging. Postharvest Biol. Technol. 56:246-

253.

Song, J., L. Fan, C. Forney, L. Campbell-Palmer and S. Fillmore. 2010. Effect of hexanal

vapor to control postharvest decay and extend shelf-life of high bush blueberry fruit

during controlled atmosphere storage. Can. J. Plant Sci. 90:359-366.

Song, J., W.E. Renderos, L.C. Palmer, C. Doucette, P.D. Hildebrand, L. Fan and C.F.

Forney. 2007. Effect of hexanal vapor on the growth of postharvest pathogens and

fruit decay. J. Food Sci. 72:108-112.

Song, L., Y. Jiang, H. Gao, C. Li, H. Liu, Y. You and J. Sun. 2006. Effects of adenosine

triphosphate on browning and quality of harvested litchi fruit. Am. J. Food Technol.

1:173-178.

Song, J., R. Leepipattwit, W. Deng. and R. Beaudry. 1996. Hexanal vapor is a natural,

metabolized fungicide: inhibition of fungal activity and enhancement of aroma

biosynthesis in apple slices. J. Am. Soc. Hortic. Sci. 121:937-942.

Stagner, D. and B.M. Popovic. 2009. Comparative study of antioxidant capacity in organs of

different Allium species. Cent. Eur. J. Biol. 4:224-228.

Steel, R.G.D., J.H. Torrie and D.A. Dickey. 1997. Principles and Procedures of Statistics: A

Biometrical Approach. 3rd ed. McGraw-Hill, New York.

Stewart, D., J. Oparka, C. Johnstone, P.P.M. Iannetta and H.V. Davies. 1999. Effect of

modified packaging (MAP) on soft fruit quality. Plant Biochem. Phytochem. 119-

124.

Sun, D., G. Liang, J. Xie, X. Lei and Y. Mo. 2010. Improved preservation effects of litchi

fruit by combining chitosan coating with ascorbic acid treatment during postharvest

storage. Afr. J. Biotechnol. 9:3272-3279.

Suttirak, W. and S. Manurakchinakorn. 2010. Potential application of ascorbic acid, citric

acid and oxalic acid for browning inhibition in fresh-cut fruits and vegetables.

Walailak J. Sci. Technol. 7:5-14.

Suwanakood, P., V. Sardsud, S. Sangchote and U. Sardsud. 2004. Microscopic observation

and pathogenicity determination of common molds on postharvest longan fruit cv.

References

176

Daw. In: The 20th Biennial Conference of the Asian Association for Biology

Education. 26-30 December 2004, Chiang Mai University, Chiang Mai, Thailand.

Tanada-Palmu, P.S. and C.R.F. Grosso. 2005. Effect of edible wheat gluten-based films and

coatings on refrigerated strawberry (Fragaria × ananassa) quality. Postharvest Biol

Technol. 36:199-208.

Terdbaramee, U., K. Ratanakhanokchai and S. Kanlayanarat. 2003. Effect of citric acid on

the control of post-harvest browning of lychee fruit under cold storage. Acta Hortic.

598:255-267.

Thavong, P., D.D. Archbold and R. Koslanund. 2010. Postharvest use of hexanal vapor and

heat treatment on longan fruit decay and consumer acceptance. Int. J. Sci. Technol.

15:4-9.

Tian, S., B. Li and Y. Xu. 2005. Effects of O2 and CO2 concentrations on physiology and

quality of litchi fruit in storage. Food Chem. 91:659-663.

Tian, S.P., Q. Fan, Y. Xu, Y. Wang and A.L. Jiang. 2001. Evaluation the use of high CO2

concentrations and cold storage to control of Monilinia fructicola on sweet cherries.


Tipton, K.F. and H.B.F. Dixon. 1983. Effects of pH on enzymes. p. 97-148. In: D.L. Purich

(ed.). Contemporary enzyme kinetics and mechanism. Academic Press, New York,

USA.

Toivonen, P.M.A. 1992. The reaction of browning in parsnips. J. Hortic. Sci. 67:547-551.

Tongdee, S.C. 1998. Postharvest technology of fresh lychee: Commercial perspective from

Thailand. South African Lychee Growers Association Yearbook. 9:37-43.

Tongdee, S.C., K.J. Scott and W.B. McGlasson. 1982. Packaging and cool storage of litchi.

CSIRO Food Res. Quart. 42:25-28.

Ullah, S., A.S. Khan, A.U. Malik and M. Shahid. 2013. Cultivar and harvest location

influence fruit softening and antioxidative activities of peach during ripening. Int. J.

Agric. Biol. 15:1059-1066.

Underhill, S.J.R. and C. Critchley. 1993. Physiological, biochemical and anatomical changes

in lychee (Litchi chinensis Sonn.) pericarp during storage. J. Hortic. Sci. 68:327-335.

References

177

Underhill, S.J.R. and C. Critchley. 1994. Anthocyanin decolourisation and its role in lychee

pericarp browning. Aust. J. Exp. Agric. 34:115-122.

Underhill, S.J.R. and C. Critchley. 1995. Cellular localization of polyphenol oxidase and

peroxidase activity in Litchi chinensis Sonn. pericarp. Aust. J. Plant Physiol. 22:627-

632.

Underhill, S.J.R. and D.H. Simons. 1993. Lychee (Litchi chinensis Sonn.) pericarp

desiccation and the importance of postharvest micro-cracking. Sci. Hortic. 54:287-

294.

Underhill, S.J.R., J. Bagshaw, A. Prasad, G. Zauberman, R. Ronen and Y. Fuchs. 1992.

The control of litchi (Litchi chinensis Sonn.) postharvest skin browning using

sulphur dioxide and low pH. Acta Hortic. 321:732-741.

Underhill, S.J.R. 1990. Lychee (Litchi chinensis Sonn.) pericarp browning. Trop. Sci.

32:305-312.

Utto, W., A.J. Mawson and J.E. Bronlund. 2008. Hexanal reduces infection of tomatoes by

Botrytis cinerea whilst maintaining quality. Postharvest Biol. Technol. 47:434-437.

Van den Berg, L. 1987. Water vapor pressure. p. 203-230. In: J. Weichmann (ed.).

Postharvest physiology of vegetables. Marcel Dekker, New York.

Waite, J.H. 1976. Calculating extinction coefficients for enzymatically produced o-quinones.

Anal. Biochem. 75:211-218.

Wall, M.M. 2006. Ascorbic acid and mineral composition of longan (Dimocarpus longan),

lychee (Litchi chinensis) and rambutan (Nephelium lappaceum) cultivars grown in

Hawaii. J. Food Compos. Anal. 19:655-663.

Wang, C.Y., H. Chen, P. Jin and H. Gao. 2010. Maintaining quality of litchi fruit with

acidified calcium sulfate. J. Agric. Food. Chem. 58:8658-8666.

Wang, Y.Y., W.Z. Hu, K. Pang, B.W. Zhu and S.D. Fan. 2007. Research progress of

browning mechanism of fruit and vegetables responding to mechanical stress. Sci.

Technol. Food Ind. 28:230-233.

Wang, X.J., S.L. Yuan, J. Wang, P. Lin, G.J. Liu, Y.R. Lu, J. Zhang, W.D. Wang and

Y.Q. Wei. 2006. Anticancer activity of litchi fruit pericarp extract against human

breast cancer in vitro and in vivo. Toxicol. Appl. Pharmacol. 215:168-178.

References

178

Wang, S.F., Z.M. Cheng, Y. Li, Y. Wang, and L.Y. Zhen. 1996. Effect of postharvest

treatments on physiology and quality of litchi and their economics. Acta Hortic.

429:503-507.

Wendehenne, D., A. Pugin, D.F. Klessig and J. Durner. 2001. Nitric oxide: comparative

synthesis and signalling in animal and plant cells. Trends Plant Sci. 6:177-183.

Wills, R., B. McGlasson, D. Graham and D. Joyce. 1998. Postharvest. An introduction to the

physiology and handling of fruit, vegetables and ornamentals. UNSW Press,

Australia. p. 143.

Wills, R.H.H., T.H. Lee, D. Graham, W.B. McGlasson, and E.G. Hall. 2004. Post-harvest.

An Introduction to the Physiology and Handling of Fruits and Vegetables. Granada

Publ. Ltd., London

Wong, K.C. 2000. Longan production in Asia. Food and Agric. Organ. United Nations.

Bangkok, Thailand. p. 44-45.

Wong, L.S., K.K. Jacobi and J.E. Giles. 1991. The influence of hot benomyl dips on the

appearance of cool stored lychee (Litchi chinensis Sonn.). Sci. Hortic. 46:245-251.

Wu, B., X. Li, H. Hu, A. Liu and W. Chen. 2011. Effect of chlorine dioxide on the control of

postharvest diseases and quality of litchi fruit. Afr. J. Biotechnol. 10: 6030-6039.

Wu, Z.X. 1995. Physiological and biochemical changes of browning in litchi fruits during

postharvest storage. MS thesis, South China Agri. Uni. Guangzhou.

Yahia, E.M., M. Contreras-Padilla and G. Gonazalez-Aguilar. 2001. Ascorbic acid content in

relation to ascorbic acid oxidase activity and polyamine content in tomato and bell

pepper fruits during development, maturation and senescence. LWT-Food Sci.

Technol. 34:452-457.

Yamaguchi, F., Y. Yoshimura, H. Nakazawa and A. Ariga. 1999. Free radical scavenging

activity of grape seed extract and antioxidants by electron spin resonance

spectrometry in an H2O2/NaOH/DMSO system. J. Agric. Food Chem. 47:2544-2548.

Yang, E., W. Lu, H. Qu, H. Lin, F. Wu, S. Yang, Y. Chen and Y. Jiang. 2009. Altered energy

status in pericarp browning of litchi fruit during storage. Pak. J. Bot. 41:2271-2279.

References

179

Yang, S., X. Su, K.N. Prasad, B. Yang, G. Cheng, Y. Chen, E. Yang and Y. Jiang. 2008.

Oxidation and peroxidation of postharvest banana fruit during softening. Pak. J. Bot.

40:2023-2029.

Yanishlieva, N.V., E. Marinova and J. Pokorny. 2006. Natural antioxidants from herbs and

spices. Eur. J. Lipid Sci. Technol. 108:776-793.

Ye, M. 2005. Advances of explant browning in plant tissue culture. J. Chongqing Technol.

Bus. Univ. 22:310-326.

Yi, H.C., S. Joo, K.H. Nam, J.S. Lee, B.G. Kang and W.T. Kim. 1999. Auxin and

brassinosteroid differentially regulate the expression of three members of the 1-

aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata

L.). Plant Mol. Biol. 41:443-454.

Yoruk, R., M.O. Balaban, M.R. Marshall and S. Yoruk. 2002. The inhibitory effect of oxalic

acid on browning of banana slices. In Annual Meeting and Food Expo, Anaheim, CA.

p. 30-18.

Yoruk, R., S. Yoruk, M.O. Balaban and M.R. Marshall. 2004. Machine vision analysis of

antibrowning potency for oxalic acid a comparative investigation on banana and

apple. J. Food Sci. 69:1365-2621.

Zauberman, G., R. Ronen, M. Akerman, A. Weksler, I. Rot and Y. Fuchs. 1991. Postharvest

retention of red colour of litchi fruit pericarp. Sci. Hortic. 47:89-97.

Zhang, D. and P.C. Quantick. 1997. Effects of chitosan coat-ing on enzymatic browning and

decay during post-harvest storage of litchi (Litchi chinensis Sonn.) fruits. Postharvest


Zhang, D.L. and P.C. Quantick. 2000. Effect of low temperature hardening on

postharvest storage of litchi fruit. Acta Hortic. 518:175-182.

Zhang, Z., X. Pang, D. Xuewu, Z. Ji and Y. Jiang. 2005. Role of peroxidase in anthocyanin

degradation in litchi fruit pericarp. Food Chem. 90:47-52.

Zhang, Z., X. Pang, J. Zuoliang and Y. Jiang. 2001a. Role of anthocyanin degradation in

litchi pericarp browning. Food Chem. 75:217-221.

References

180

Zhang, Z. S., R.Q. Li and J.B. Wang. 2001b. Effects of oxalate treatment on the membrane

permeability and calcium distribution in pepper leaves under heat stress. Acta

Phytophysiol. Sinica. 27:109-113.

Zhao, M.M., B. Yang, J.S. Wang, Y. Liu, L.M. Yu and Y.M. Jiang. 2007.

Immunomodulatory and anticancer activities of flavonoids extracted from litchi

(Litchi chinensis Sonn.) pericarp. Int. Immunopharmacol. 7:162-166.

Zhao, M.M., B. Yang, J.S. Wang, B.Z. Li and Y.M. Jiang. 2006. Identification of the major

flavonoids from pericarp tissues of lychee fruit in relation to their antioxidant

activities. Food Chem. 98:539-544.

Zheng, G.Y., R.L. Zhao and X. Peng. 1999. Oxalate introduces muskmelon resistance to

WMV-2. Chinese Sci. Bull. 44:1059-1062.

Zheng, H., S.S. Fang, H.Q. Lou, Y. Chen, L.L. Jiang and H.F. Lu. 2011. Neural network

prediction of ascorbic acid degradation in green asparagus during thermal treatments.

Expert Syst. Appl. 38:5591-5602.

Zheng, X. and S. Tian. 2006. Effect of oxalic acid on control of postharvest browning of

litchi fruit. Food Chem. 96:519-523.

Zheng, X., S. Tian, M.J. Gidleyc, H. Yued and B. Lia. 2007a. Effects of exogenous oxalic

acid on ripening and decay incidence in mango fruit during storage at room

temperature. Postharvest Biol. Technol. 45:281-284.

Zheng, X., S. Tian, X. Meng and B. Li. 2007b. Physiological and biochemical responses in

peach fruit to oxalic acid treatment during storage at room temperature. Food Chem.

104:156-162.

prr.hec.gov.pkprr.hec.gov.pk/jspui/bitstream/123456789/6996/1/muhammad_shafique... · iii to the...

Documents