Advances in Postharvest

Fruit and Vegetable

Technology

EDITED BY

Ron B.H. Wills • John Brett Golding

Advances in Postharvest
Fruit and Vegetable
Technology
 Contemporary Food Engineering
Series Editor
Professor Da-Wen Sun, Director
Food Refrigeration & Computerized Food Technology
National University of Ireland, Dublin
(University College Dublin)
Dublin, Ireland
http://www.ucd.ie/sun/

Advances in Postharvest Fruit and Vegetable Technology,

edited by Ron B.H. Wills and John Brett Golding (2015)
Engineering Aspects of Food Emulsification and Homogenization,
edited by Marilyn Rayner and Petr Dejmek (2015)
Handbook of Food Processing and Engineering, Volume II: Food Process
Engineering, edited by Theodoros Varzakas and Constantina Tzia (2014)
Handbook of Food Processing and Engineering, Volume I: Food Engineering
Fundamentals, edited by Theodoros Varzakas and Constantina Tzia (2014)
Juice Processing: Quality, Safety and Value-Added Opportunities, edited by
Víctor Falguera and Albert Ibarz (2014)
Engineering Aspects of Food Biotechnology, edited by José A. Teixeira
and António A. Vicente (2013)
Engineering Aspects of Cereal and Cereal-Based Products, edited by
Raquel de Pinho Ferreira Guiné and Paula Maria dos Reis Correia (2013)
Fermentation Processes Engineering in the Food Industry, edited by
Carlos Ricardo Soccol, Ashok Pandey, and Christian Larroche (2013)
Modified Atmosphere and Active Packaging Technologies, edited by
Ioannis Arvanitoyannis (2012)
Advances in Fruit Processing Technologies, edited by Sueli Rodrigues and
Fabiano Andre Narciso Fernandes (2012)
Biopolymer Engineering in Food Processing, edited by Vânia Regina Nicoletti
Telis (2012)
Operations in Food Refrigeration, edited by Rodolfo H. Mascheroni (2012)
Thermal Food Processing: New Technologies and Quality Issues, Second
Edition, edited by Da-Wen Sun (2012)
Physical Properties of Foods: Novel Measurement Techniques and
Applications, edited by Ignacio Arana (2012)
Handbook of Frozen Food Processing and Packaging, Second Edition,
edited by Da-Wen Sun (2011)
Advances in Food Extrusion Technology, edited by Medeni Maskan and
Aylin Altan (2011)
Enhancing Extraction Processes in the Food Industry, edited by Nikolai Lebovka,
Eugene Vorobiev, and Farid Chemat (2011)
Emerging Technologies for Food Quality and Food Safety Evaluation,
edited by Yong-Jin Cho and Sukwon Kang (2011)
Food Process Engineering Operations, edited by George D. Saravacos and
Zacharias B. Maroulis (2011)
Biosensors in Food Processing, Safety, and Quality Control, edited by Mehmet
Mutlu (2011)
Physicochemical Aspects of Food Engineering and Processing, edited by
Sakamon Devahastin (2010)
Infrared Heating for Food and Agricultural Processing, edited by Zhongli Pan
and Griffiths Gregory Atungulu (2010)
Mathematical Modeling of Food Processing, edited by Mohammed M. Farid (2009)
Engineering Aspects of Milk and Dairy Products, edited by Jane Sélia dos Reis
Coimbra and José A. Teixeira (2009)
Innovation in Food Engineering: New Techniques and Products, edited by Maria
Laura Passos and Claudio P. Ribeiro (2009)
Processing Effects on Safety and Quality of Foods, edited by Enrique Ortega-
Rivas (2009)
Engineering Aspects of Thermal Food Processing, edited by Ricardo Simpson
(2009)
Ultraviolet Light in Food Technology: Principles and Applications,
Tatiana N. Koutchma, Larry J. Forney, and Carmen I. Moraru (2009)
Advances in Deep-Fat Frying of Foods, edited by Serpil Sahin and Servet Gülüm
Sumnu (2009)
Extracting Bioactive Compounds for Food Products: Theory and Applications,
edited by M. Angela A. Meireles (2009)
Advances in Food Dehydration, edited by Cristina Ratti (2009)
Optimization in Food Engineering, edited by Ferruh Erdoǧdu (2009)
Optical Monitoring of Fresh and Processed Agricultural Crops, edited by
Manuela Zude (2009)
Food Engineering Aspects of Baking Sweet Goods, edited by Servet Gülüm
Sumnu and Serpil Sahin (2008)
Computational Fluid Dynamics in Food Processing, edited by Da-Wen Sun
(2007)
Advances in Postharvest
Fruit and Vegetable
Technology
EDITED BY
Ron B.H. Wills • John Brett Golding

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Taylor & Francis Group, an informa business
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Contents
Series Preface.............................................................................................................ix
Series Editor...............................................................................................................xi
Preface.................................................................................................................... xiii
Acknowledgment...................................................................................................... xv
Editors.....................................................................................................................xvii
Contributors.............................................................................................................xix

Chapter 1 Postharvest Technology Experimentation: Solutions

to Common Problems............................................................................ 1
John Brett Golding and Lorraine Spohr

Chapter 2 Recent Research on Calcium and Postharvest Behavior..................... 19

Babak Madani and Charles F. Forney

Chapter 3 Nondestructive Assessment of Fruit Quality...................................... 39

Kerry Walsh

Chapter 4 Biological Control of Postharvest Diseases........................................ 65

Giuseppe Lima, Simona Marianna Sanzani, Filippo De Curtis,
and Antonio Ippolito

Chapter 5 Physical and Chemical Control of Postharvest Diseases.................... 89

Alice Spadoni, Fiorella Neri, and Marta Mari

Chapter 6 Advances in the Use of 1-MCP......................................................... 117

Chris B. Watkins

Chapter 7 Advances in Edible Coatings............................................................ 147

María Serrano, Domingo Martínez-Romero, Pedro J. Zapata,
Fabián Guillén, Juan M. Valverde, Huertas M. Díaz-Mula,
Salvador Castillo, and Daniel Valero

Chapter 8 Low Ethylene Technology in Non-Optimal Storage Temperatures.......167

Ron B.H. Wills

vii
viii Contents

Chapter 9 Potential of Nitric Oxide as a Postharvest Technology..................... 191

Ron B.H. Wills

Chapter 10 Methyl Jasmonate in Postharvest...................................................... 211

Ahmad Sattar Khan, Zora Singh, and Sajid Ali

Chapter 11 Postharvest Oxidative Stress in Fresh Fruits..................................... 237

Sukhvinder Pal Singh

Chapter 12 Advances in Postharvest Maintenance of Flavor and

Phytochemicals................................................................................. 261
Jun Song

Chapter 13 Metabolomics Tools for Postharvest Quality and Safety

of Fresh Produce................................................................................ 285
Sukhvinder Pal Singh

Chapter 14 Recent Developments in Proteomic Analysis of Fruits....................309

Jun Song

Chapter 15 Organic Postharvest Technology....................................................... 331

Apiradee Uthairatanakij and Pongphen Jitareerat

Chapter 16 Modeling in Postharvest Horticulture............................................... 347

Maarten L.A.T.M. Hertog and Bart M. Nicolaï
Series Preface
CONTEMPORARY FOOD ENGINEERING
Food engineering is the multidisciplinary field of applied physical sciences combined
with the knowledge of product properties. Food engineers provide the technological
knowledge transfer essential to the cost-effective production and commercialization
of food products and services. In particular, food engineers develop and design pro-
cesses and equipment to convert raw agricultural materials and ingredients into safe,
convenient, and nutritious consumer food products. However, food engineering top-
ics are continuously undergoing changes to meet diverse consumer demands, and the
subject is being rapidly developed to reflect market needs.
In the development of food engineering, one of the many challenges is to employ
modern tools and knowledge, such as computational materials science and nano-
technology, to develop new products and processes. Simultaneously, improving food
quality, safety, and security continues to be critical issues in food engineering ­studies.
New packaging materials and techniques are being developed to provide more pro-
tection to foods, and novel preservation technologies are emerging to enhance food
security and defense. Additionally, process control and automation regularly appear
among the top priorities identified in food engineering. Advanced monitoring and
control systems are developed to facilitate automation and flexible food manufac-
turing processes. Furthermore, energy-saving and minimization of environmental
problems continue to be important food engineering issues, and significant progress
is being made in waste management, efficient utilization of energy, and reduction of
effluents and emissions in food production.
The Contemporary Food Engineering Series, consisting of edited books, attempts
to address some of the recent developments in food engineering. The series covers
advances in classical unit operations in engineering applied to food manufacturing
as well as topics such as progress in the transport and storage of liquid and solid
foods; heating, chilling, and freezing of foods; mass transfer in foods; chemical and
biochemical aspects of food engineering and the use of kinetic analysis; dehydration,
thermal processing, non-thermal processing, extrusion, liquid food concentration,
membrane processes, and applications of membranes in food processing; shelf-life
and electronic indicators in inventory management; sustainable technologies in food
processing; and packaging, cleaning, and sanitation. These books are aimed at pro-
fessional food scientists, academics researching food engineering problems, and
graduate-level students.
The editors of these books are leading engineers and scientists from different
parts of the world. All the editors were asked to present their books to address the
market’s needs and pinpoint cutting-edge technologies in food engineering.
All contributions are written by internationally renowned experts who have
both academic and professional credentials. All authors have attempted to provide

ix
x Series Preface

critical, comprehensive, and readily accessible information on the art and science
of a r­elevant topic in each chapter, with reference lists for further information.
Therefore, each book can serve as an essential reference source to students and
researchers in universities and research institutions.

Da-Wen Sun
Series Editor
Series Editor
Prof. Da-Wen Sun, born in southern China, is
a global authority in food engineering research
and education; he is a member of the Royal Irish
Academy (RIA), which is the highest academic
honor in Ireland; he is also a member of Academia
Europaea (The Academy of Europe) and a fellow
of the International Academy of Food Science and
Technology. He has contributed significantly to the
field of food engineering as a researcher, as an aca-
demic authority, and as an educator.
His main research activities include cooling,
drying, and refrigeration processes and systems,
quality and safety of food products, bioprocess simulation and optimization, and
computer vision/image processing and hyperspectral imaging technologies. His many
scholarly works have become standard reference materials for researchers, espe-
cially in the areas of computer vision, computational fluid dynamics modeling, vac-
uum cooling, and related subjects. Results of his work have been published in over
800 papers, including more than 390 peer-reviewed journal-papers (Web of Science
h-index = 62). He has also edited 14 authoritative books. According to Thomson
Scientific’s Essential Science IndicatorsSM, based on data derived over a period of
10 years from Web of Science, there are about 4,500 scientists who are among the
top 1% of the most cited scientists in the category of agriculture sciences; for many
years, Professor Sun has consistently been ranked among the top 50 scientists in the
world (he was at 25th position in March 2015, and at 1st position if ranking is based
on “Hot Papers,” and in 2nd position if ranking is based on “Top Papers” or “Highly
Cited Papers”).
He received a first class BSc honors and MSc in mechanical engineering, and
a PhD in chemical engineering in China before working in various universities in
Europe. He became the first Chinese national to be permanently employed in an
Irish university when he was appointed college lecturer at the National University of
Ireland, Dublin (University College Dublin, UCD), in 1995, and was then progres-
sively promoted in the shortest possible time to senior lecturer, associate professor,
and full professor. Dr. Sun is now a professor of food and biosystems engineering
and the director of the UCD Food Refrigeration and Computerized Food Technology
Research Group.
As a leading educator in food engineering, Professor Sun has trained many PhD
students, who have made their own contributions to the industry and academia. He
has also frequently delivered lectures on advances in food engineering at academic
institutions worldwide, and delivered keynote speeches at international conferences.
As a recognized authority in food engineering, he has been conferred adjunct/­
visiting/consulting professorships from 10 top universities in China, including

xi
xii Series Editor

Zhejiang University, Shanghai Jiaotong University, Harbin Institute of Technology,

China Agricultural University, South China University of Technology, and Jiangnan
University. In recognition of his significant contribution to food ­engineering ­worldwide
and for his outstanding leadership in the field, the International Commission of
Agricultural and Biosystems Engineering (CIGR) awarded him the “CIGR Merit
Award” in 2000, and again in 2006, the Institution of Mechanical Engineers based
in the United Kingdom named him “Food Engineer of the Year 2004.” In 2008, he
was awarded the “CIGR Recognition Award” in honor of his distinguished achieve-
ments as one of the top 1% among agricultural engineering scientists in the world.
In 2007, he was presented with the only “AFST(I) Fellow Award” given in that year
by the Association of Food Scientists and Technologists (India), and in 2010, he was
presented with the “CIGR Fellow Award”; the title of Fellow is the highest honor at
CIGR and is conferred to individuals who have made sustained, outstanding contri-
butions worldwide. In March 2013, he was presented with the “You Bring Charm
to the World” Award by Hong Kong-based Phoenix Satellite Television with other
award recipients including the 2012 Nobel Laureate in Literature and the Chinese
Astronaut Team for Shenzhou IX Spaceship. In July 2013, he received the “Frozen
Food Foundation Freezing Research Award” from the International Association for
Food Protection (IAFP) for his significant contributions to enhancing the field of
food-freezing technologies. This is the first time that this prestigious award was pre-
sented to a scientist outside the United States.
He is a fellow of the Institution of Agricultural Engineers and a fellow of Engi­neers
Ireland (the Institution of Engineers of Ireland). He is editor-in-chief of Food and
Bioprocess Technology—An International Journal (2012 Impact Factor = 4.115),
former editor of Journal of Food Engineering (Elsevier), and a member of the edi-
torial boards for a number of international journals, including the Journal of Food
Process Engineering, Journal of Food Measurement and Characterization, and
Polish Journal of Food and Nutritional Sciences. He is also a chartered engineer.
On May 28, 2010, he was awarded membership in the RIA, which is the highest
honor that can be attained by scholars and scientists working in Ireland. At the 51st
CIGR General Assembly held during the CIGR World Congress in Quebec City,
Canada, on June 13–17, 2010, he was elected incoming president of CIGR, became
CIGR president in 2013–2014, and is now CIGR past president. On September 20,
2011, he was elected to Academia Europaea (The Academy of Europe), which is
functioning as the European Academy of Humanities, Letters and Sciences, and
is one of the most prestigious academies in the world; election to the Academia
Europaea represents the highest academic distinction.
Preface
The aim of postharvest technology is to transfer fruit and vegetables from the farm to
consumers without loss of quantity and quality. Great advances have occurred since
the 1950s, when there was a seasonal availability of most produce with consumption
close to production areas and where quality was secondary to availability. In the
intervening years, the horticulture industry has advanced to a situation where there
is now year-round availability for many commodities, which is supported by a sub-
stantial international trade. In addition, consumer expectations of fresh produce and
quality have substantially changed in the last few decades. A greater diversity and
quantity of produce are marketed but high quality is paramount, although there are
various concepts of what constitutes quality. This development was made possible
by the introduction of a range of technologies but the major technological impacts
can be ascribed to

• Cool-chain management, where a controlled temperature environment is

maintained from farm storage to domestic consumption to inhibit ripening
and senescence.
• Availability of synthetic chemicals, mainly to inhibit the growth of diseases
and pests.

However, changes in the use of these technologies are being driven by evolving
consumer expectations, community attitudes, and economic pressures on the indus-
try. Important among these are the

• Desire by the international community to minimize environmental degra-

dation; this has led to a range of government policy initiatives to stop the
use of ozone depleting substances and reduce greenhouse gas emissions.
• Growing consumer aversion to the presence of synthetic chemicals in foods
and a desire for what is perceived to be more “natural” and “safe” foods,
which effectively translates into reduced usage of synthetic chemicals.
• Use of smarter, more efficient technologies to assess and manage quality.

This volume examines a range of recently developed technologies and systems

that will assist the horticulture industry in being more environmentally sustainable
and economically competitive, in minimizing postharvest quality loss, and in the use
of biomolecular tools in research and applications.

xiii
Acknowledgment
The editors wish to acknowledge the mentoring role of Dr. Barry McGlasson, first
as the supervisor of their doctoral programs, but also as the source of wise counsel-
ing and friendship over many years. Dr. McGlasson’s significant scientific advances
to knowledge during his 50-year career have profoundly influenced the postharvest
community around the world. He has an infectious enthusiasm for grasping new
scientific tools that lead to a better understanding of the physiology and biochem-
istry of postharvest systems, and then finding innovative ways of translating this
knowledge into practical outcomes. Dr. McGlasson has a unique ability to inspire
his students and colleagues to be creative thinkers not only in postharvest but in
life generally. He is passionate about the importance of horticulture and has been
a strong and enthusiastic advocate for horticultural science, and the future of hor-
ticultural education. Dr. McGlasson’s contributions to horticulture have been rec-
ognized in both Australia and internationally. He is a fellow of the International
Society of Horticultural Science. His continuing support and lifelong friendship are
greatly valued.

xv
Editors
Dr. Ron B.H. Wills is an emeritus professor in the School of Environmental and
Life Sciences at the University of Newcastle, Australia. He has 50 years experi-
ence in many aspects of postharvest horticulture and has published 300 research
papers. He has held government, university, and industry positions in Australia and
New Zealand, and has consulted for government and international agencies on post­
harvest development projects throughout Asia and the South Pacific.

Dr. John Brett Golding works for the New South Wales Department of Primary
Industries (Australia), where he conducts applied postharvest and market access
research for a range of horticulture industries. He has had extensive experience in
applied food science, postharvest physiology, and supply chain work. Dr. Golding
also holds a conjoint position within the School of Environmental and Life Sciences
at the University of Newcastle in Australia.

xvii
Contributors
Sajid Ali Antonio Ippolito
Institute of Horticultural Sciences Department of Soil, Plant and Food
University of Agriculture Sciences
Punjab, Pakistan University of Bari Aldo Moro
Bari, Italy
Salvador Castillo
Department of Food Technology Pongphen Jitareerat
Miguel Hernández University Division of Postharvest Technology
Alicante, Spain School of Bioresouces and Technology
King Mongkut’s University of
Filippo De Curtis Technology Thonburi
Department of Agricultural Bangkok, Thailand
Environmental and Food Sciences
University of Molise
Campobasso, Italy Ahmad Sattar Khan
Institute of Horticultural Sciences
Huertas M. Díaz-Mula University of Agriculture
Department of Food Technology Punjab, Pakistan
Miguel Hernández University
Alicante, Spain Giuseppe Lima
Department of Agricultural
Charles F. Forney Environmental and Food Sciences
Agriculture and Agri-Food Canada University of Molise
Atlantic Food and Horticulture Campobasso, Italy
Research Centre
Kentville, Nova Scotia, Canada Babak Madani
Department of Crop Science
John Brett Golding Universiti Putra Malaysia
New South Wales Department of Selangor, Malaysia
Primary Industries
New South Wales, Australia
Marta Mari
Fabián Guillén CRIOF, DipSA
Department of Food Technology University of Bologna
Miguel Hernández University Bologna, Italy
Alicante, Spain
Domingo Martínez-Romero
Maarten L.A.T.M. Hertog Department of Food Technology
BIOSYST-MeBioS, KU Leuven Miguel Hernández University
Leuven, Belgium Alicante, Spain

xix
xx Contributors

Fiorella Neri Lorraine Spohr

CRIOF, DipSA New South Wales Department of
University of Bologna Primary Industries
Bologna, Italy New South Wales, Australia

Bart M. Nicolaï Apiradee Uthairatanakij

BIOSYST-MeBioS, KU Leuven Division of Postharvest Technology
and School of Bioresouces and
VCBT Technology
Leuven, Belgium King Mongkut’s University of
Technology Thonburi
Simona Marianna Sanzani Bangkok, Thailand
Department of Soil, Plant and Food
Sciences Daniel Valero
University of Bari Aldo Moro Department of Food Technology
Bari, Italy Miguel Hernández University
Alicante, Spain
María Serrano
Department of Applied Biology Juan M. Valverde
Miguel Hernández University Department of Food Technology
Alicante, Spain Miguel Hernández University
Alicante, Spain
Sukhvinder Pal Singh
New South Wales Department of
Kerry Walsh
Primary Industries
Central Queensland University
New South Wales, Australia
Queensland, Australia
Zora Singh
Department of Environment and Chris B. Watkins
Agriculture Department of Horticulture
Curtin University Cornell University
Western Australia, Australia Ithaca, New York

Jun Song Ron B.H. Wills

Agriculture and Agri-Food Canada School of Environmental and Life
Atlantic Food and Horticulture Sciences
Research Centre University of Newcastle
Kentville, Nova Scotia, Canada New South Wales, Australia

Alice Spadoni Pedro J. Zapata

CRIOF, DipSA Department of Food Technology
University of Bologna Miguel Hernández University
Bologna, Italy Alicante, Spain
 1 Postharvest Technology
Experimentation
Solutions to Common
Problems
John Brett Golding and Lorraine Spohr

CONTENTS
1.1 Introduction.......................................................................................................1
1.2 Solutions for Common Problems in Postharvest Technology
Experimentation................................................................................................2
1.2.1 Acknowledging the Literature...............................................................2
1.2.2 Ignoring Variability...............................................................................3
1.2.3 Unknown History of Experimental Material........................................3
1.2.4 Failing to Acknowledge Scope of Inference..........................................6
1.2.5 Failing to Use a Good Experimental Design......................................... 7
1.2.6 Poor Description of Experimental Design.............................................7
1.2.7 Understanding Replication....................................................................7
1.2.8 Pseudoreplication...................................................................................8
1.2.9 Sub-Sampling...................................................................................... 12
1.2.10 Incorrect Statistical Analysis............................................................... 12
1.2.11 Reporting Statistical Analysis Methods and Results........................... 14
1.2.12 Statistical Significance versus Biological Significance of Results......... 15
1.3 Conclusions...................................................................................................... 16
Acknowledgment...................................................................................................... 16
References................................................................................................................. 16

1.1 INTRODUCTION
Postharvest research aims to improve the availability and quality of horticultural
produce by developing new ideas and assessing these ideas by conducting experi-
ments and following the scientific method. The scientific method requires a well-
defined and reported experiment, which then ensures the validity and defines the
scope of any subsequent conclusions.
Sound statistical design and analysis of postharvest experiments are essential to
the interpretation of results and inferences that can be drawn. Many of the tools that

1
2 Advances in Postharvest Fruit and Vegetable Technology

postharvest researchers use to design, conduct, and analyze experiments are some-
times misused, and consequently find their way into the literature. Poorly designed,
and analysed experiments often lead to biased results and conclusions. This chapter
is a collection of common problems encountered in postharvest technology research
and suggested solutions to these problems.
After reviewing papers from three international postharvest journals published
in 2014, 50 papers were studied in detail. We found 56% of these papers had no or
poor descriptions of the experimental design. This poor description of the design
makes it difficult to know how the experiment was setup and conducted. While
minute details are often provided, describing the manufacturer of reagents and
equipment, comparatively less detail is provided in describing the experimental
design.
In this survey of recently published postharvest literature, we also found that the
majority of papers surveyed had poor statistical analysis descriptions and presenta-
tion of the results. We found that 62% of the papers had either no or poor descrip-
tions of the statistical methods, while 56% had poor presentation of the results.
From this survey, we identified twelve common problems in postharvest technology
research:

1. Acknowledging the literature

2. Ignoring variability
3. Unknown history of experimental material
4. Failing to acknowledge scope of inference
5. Failing to use a good experimental design
6. Poor description of experimental design
7. Understanding replication
8. Pseudoreplication
9. Sub-sampling
10. Incorrect statistical analysis, for example, using ANOVA (analysis of vari-
ance), regardless of data type and not checking the assumptions
11. Reporting of statistical analysis methods and results
12. Statistical significance versus biological significance of results

1.2 SOLUTIONS FOR COMMON PROBLEMS IN

POSTHARVEST TECHNOLOGY EXPERIMENTATION
This section discusses the issues and problems that were identified in the literature
survey (Section 1.1) and provides potential solutions. In addition, Chapter 16 further
explores some of these issues in the context of modeling.

1.2.1  Acknowledging the Literature

As a first step in planning a postharvest experiment, it is essential to adequately
acknowledge previously published results by thoroughly exploring the literature.
This sounds like a menial task, but there have been many well-meaning experiments
that have failed to access the literature. To conduct a thorough literature review,
Postharvest Technology Experimentation 3

begin an online keyword search with a library database search engine, searching
over all possible years. Repeat this search with at least one other search engine.
Read the relevant papers and search the references from those papers. Attempts
should be made to access book chapters, reviews, and conference proceedings in
your literature review. These sources may not be easily located in the searchable
electronic journals. This suggestion may appear trite to experienced researchers;
however, there are many relevant older studies that could easily be overlooked if only
databases are used.

1.2.2 Ignoring Variability
Variation is a natural feature of biological systems. Fundamental to the design and
analysis of experiments is the appreciation and management of variability. The
variation in produce quality parameters, such as soluble solids content (SSC) lev-
els, is an inherent part of horticulture and postharvest science. Acknowledgment
and management of produce variation, such as where it comes from, why it occurs
and how to manage it, is crucial to good postharvest science. Identifying the
sources of variability and taking them into account when designing postharvest
experiments is essential, as undetected bias can contribute to spurious treatment
effects.

1.2.3 Unknown History of Experimental Material

Finding out as much as possible about the history of your experimental material will
assist in experimentation. Produce variation comes from a wide range of interacting
pre- and postharvest factors. Preharvest factors such as cultivar, rootstock, soil type,
irrigation and fertilizer regime, and fruit maturity at harvest, can affect postharvest
storage behavior.
For example rootstocks can significantly affect maturity and SSC accumulation
(Stenzel et  al., 2006). It is easy to ignore fruit maturity and assume all fruit are
“commercially mature,” based on background color or fruit blush since color may
not necessarily reflect physiological maturity. Relying on “commercial maturity” is
a common practice in postharvest experimentation but can contribute to the often
contradictory results sometimes found in the literature. For example, the effect of
1-methylcyclopropene (1-MCP) application to peaches has been reported to have
varying effects on the development of chilling-related injury. Many peach studies
with 1-MCP have been conducted using fruit with poorly defined maturity at har-
vest. However, in a clearly defined fruit-maturity study, 1-MCP treatment increased
the severity of chilling-related injury in early maturity peach fruit but had either no
effect on commercially mature fruit or even slightly delayed chilling-related injury
(Jajo et  al., 2012). This illustrates the importance of knowing fruit maturity and
adequately defining the physiology of the fruit before and after storage. The concept
of biological variation is further explored in Chapter 16.
Ripening and climacteric behaviors, chilling-sensitivities, and storage potential,
can all vary between different cultivars of the same produce type. It is therefore
important to adequately report the cultivar (or variety) used in an experiment.
4 Advances in Postharvest Fruit and Vegetable Technology

Sourcing of fruit from the general market is a convenient method to obtain pro-
duce for postharvest experiments but is not recommended, unless the aim of the
research is specifically to describe variability in the marketplace. Produce pur-
chased from the market floor may not have enough relevant history. For example,
fruit sourced from the market is of unknown biological maturity and origin and may
have been harvested several days or even weeks previously. In addition, the storage
conditions during the time spent in the market chain are unknown. The produce has
been exposed to unknown temperature conditions and ethylene levels. To have con-
fidence in the experimental results, it is important to have a thorough knowledge of
the background of the produce to be used in the experiment.
In addition, the application of plant growth regulators in the orchard can have a
significant impact on ripening behavior during postharvest storage. For example,
it is essential to know if your produce had been treated with orchard treatments
such as aminoethoxyvinylglycine (AVG). This compound is known to competitively
inhibit the activity of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase
which is the rate limiting enzyme in the ethylene biosynthetic pathway. AVG is gen-
erally applied 28 days before harvest and is used to extend the harvest period and
increase the storage-life of the apples. Knowing if your produce has been treated
with such chemicals is essential to properly conduct and interpret postharvest stor-
age experiments.
Produce variation occurs at many different levels:

• Within a single fruit: Physiological differences may be observed within a

single fruit. An example of this is the red blush (anthocyanin) associated
with the sun-exposed side of apple fruit. In addition, temperature differ-
ences across the fruit, from exposed to unexposed sides, can exceed 10°C
(Woolf et al., 1999). This means that the sun-exposed side of the fruit may
have a different physiology at certain times of the day and this may have
consequences for the fruit’s postharvest behavior. There may also be sig-
nificant gradients in SSC levels within some fruits. In extreme cases, the
SSC may vary by up to 4% Brix levels within a single peach fruit (Golding
et al., 2006). This variability has important consequences when sampling
and measuring a fruit SSC.
• Within a tree: There can be large differences in produce quality from the
same tree. Variation in fruit-size and dry matter content, within a tree and
within individual branches, has been related to heterogeneity in light inter-
ception, the fruit’s proximity to carbohydrate sources, fruit-load, and inter-
fruit competition (Lopresti et  al., 2014). The SSC levels within a single
nectarine tree at harvest are presented in Figure 1.1. Although the average
SSC level of all fruit was 14.2% Brix, the range of fruit SSC was 10%–21%
Brix of fruit on the same tree at “commercial” harvest. Harvesting and
using all the fruit from this tree in a postharvest experiment to investi-
gate the effect of postharvest treatments on SSC would be challenging. In
general, the top half of the tree had higher levels of fruit SSC compared
to the bottom half. Even within the top of the trees, the average SSC of
Postharvest Technology Experimentation 5

17.2 15.5 15.0 14.5

19.6 16.0
16.3 2.9 m

14.5 13.2 16.4

13.5 12.9

16.0
14.4 12.0 2.2 m
13.1
14.9 13.5 14.2
15.0 11.9
12.5 14.1
17.3
14.0
15.3 15.3 1.7 m
12.3
15.5 12.9 15.5
12.9 14.6
12.6 13.3
13.1
14.2
14.1 13.8
10.8 14.8
16.4 15.6
15.5
12.5
15.5
13.2 14.0
13.6
12.1

SSC (%)
East West
<12 12–14 14–16 >16

FIGURE 1.1  Schematic version of Arctic Snow nectarine tree showing average SSC% on
each fruiting lateral. (From Golding JB. 2008. Scoping study to identify and quantify factors
to improve summer-fruit quality and consistency. Horticulture Australia Project SF 06013.
ISBN 0 7241 1909 7. p 161. With permission.)

some fruiting laterals was lower and not consistent with their position on
the branches. Not only can fruit SSC vary within a single tree, numerous
other physiological and biological factors can vary and affect postharvest
behavior. For example, Magwaza et  al. (2013) showed that variations in
microclimatic conditions inside a citrus-tree canopy during the growing
season, affect the biochemical profile of the fruit rind, which, in turn, influ-
ences fruit response to postharvest stresses associated with senescence and
susceptibility to rind breakdown in mandarin fruit.
• Within a row and within an orchard or field: Orchard and field management
practices (nutrition, irrigation) and growing conditions (row orientation,
elevation) can affect the final produce growth, quality, and physiological
maturity. For example, nitrogen nutrition has been shown to affect the level
of storage disorders in numerous crops, such as apples (Little and Holmes,
2000). Although there may be similar grower management practices (such
as same fertilizer and irrigation management) in an entire row/orchard, there
may be different soils or elevations, which may affect plant growth and fruit
maturity and quality at harvest and subsequent postharvest storage behavior.
• Within growing regions and between different growing regions/countries:
The variation in produce quality and postharvest behavior can be further
exaggerated by different grower practices (irrigation, fertilizer, thinning,
etc.), rootstocks and growing conditions between geographical regions. All
6 Advances in Postharvest Fruit and Vegetable Technology

these differences in produce quality may affect postharvest storage behav-

ior in different ways and underline the need to appreciate and manage pro-
duce variability. Experiments claiming to show regional effects of quality
based on a single orchard should be interpreted with caution. Similar care
should be taken when comparing production methods such as organic ver-
sus conventional on postharvest quality.
• Variation in the postharvest environment and unknown sources of varia-
tion: Examples of this type of variability can occur within the postharvest
cool room or shipping container, where the delivery air from the refrig-
eration unit can be substantially cooler than the return air. While this may
be acknowledged, the accumulated differences in storage temperature
over the whole life of the experiment can be significant. If the experiment
is not designed correctly, treatments may be confounded with sources of
variability. For example, if an entire treatment was stored in the return-
air-stream, this treatment would be subject to higher storage tempera-
ture for the entire storage time, compared to those stored in the delivery
air. The effect of treatment temperatures within a pallet stack should also
be managed. In addition to temperature, it is also important to manage
and record relative humidity (RH). RH is directly responsible for a wide
range of postharvest issues such as weight loss, firmness, and incidence
of decay, and can regularly vary by up to 35% between storage environ-
ments. Therefore, it is important to control and accurately monitor RH in
the storage environment.

It is also important to appreciate and manage other sources of potential contami-

nation within a cool room such as ethylene, where there may also be other com-
modities or experiments in the same cool room. Some commodities, such as green
beans, are sensitive to low ethylene levels (0.01 μL/L), and these levels of ethylene
have been shown to accumulate in cool rooms where other high-ethylene-producing
commodities (such as apples) are also stored.

1.2.4 Failing to Acknowledge Scope of Inference

It is a common practice to exclude variability from a sample population when select-
ing fruit for an experiment, which results in a uniform sample population at the start
of the experiment. Whilst this does eliminate “outliers,” the use of limited sample
populations also limits the inference and conclusions. Authors and readers often
unintentionally extend inference of these results to the entire population instead of
just to the uniform part chosen for the study. To ensure that the results of postharvest
experiments are widely applicable, it is important to sample from broad popula-
tions. The scope of inference provides the context in which the results of an experi-
ment apply to a broader population of fruit. The scope of inference should be clearly
defined and applied in each study. For example, a fruit-softening experiment that
used only uniformly large-sized peaches, produced results that are applicable only to
similarly sized fruit of that variety. Further extension to other peach types and sizes
need to be conducted to confirm these findings.
Postharvest Technology Experimentation 7

1.2.5 Failing to Use a Good Experimental Design

As stated by Rubin (2008), “Design Trumps Analysis.” A good experimental design
minimizes bias and incorporates known sources of variation, which may affect the
response of interest. When sources of variation have been identified, a randomized
complete block (RCB) design is often used. A “block” is a group of experimental
units that are as similar as possible and/or experience similar conditions. The word
“complete” refers to the condition that the whole (or complete) set of treatments
being evaluated is represented within each block. It is important to recognize and
correctly use an RCB design. With the correct use of an RCB, we can incorporate
potential variability between farms or orchards by forming the blocks based on fruit
source (where a block represents a farm or orchard), and allocating a complete set of
treatments to the experimental units within each block. In the subsequent statistical
analysis, comparing treatment means differences between farms will cancel out, and
unexplained (residual) variation is reduced.
In the laboratory setting, an example of efficient use of an RCB design is when
blocks are based on a time order. For example, when assessing produce quality after
a storage experiment instead of assessing all of “Treatment 1” at once, then assessing
“Treatment 2,” it is better to use blocks. This is where a complete set of treatments
(Block 1) is assessed in the morning and another (Block 2) at mid-day and a third
(Block 3) in the afternoon. Any bias due to operator (e.g., fatigue) or time of day will
be accounted for in the subsequent statistical analysis by the blocking structure.
Many authors often state that they use an RCB when they have actually used a
completely randomized design (CRD). A CRD is recommended when no known
sources of variation—that may affect the experiment—can be identified, and the
experimental material is homogeneous.

1.2.6 Poor Description of Experimental Design

A clear description of the type of experimental design, the number of replicates,
a ­definition of the experimental unit, and type of statistical analysis method employed,
is essential. This should provide enough detail so that someone else could repeat the
experiment at another time and place, without the need to make any assumptions
about how the experiment was setup or conducted.
In any postharvest experiment, it is important to clearly define the experimen-
tal unit for each treatment. The experimental unit is the physical unit to which a
treatment is independently applied, usually through a randomization process. An
experimental unit may be an individual (a single apple) or a group (five apples),
depending on the experimental protocol. The identification of experimental units,
the independence of the experimental unit and the consequent scope of inference
are all important concepts to understand and apply to all postharvest experiments.

1.2.7 Understanding Replication
True replication occurs when the same treatment is independently applied to a num-
ber of experimental units. Replicating (or repeating the application of) treatments
8 Advances in Postharvest Fruit and Vegetable Technology

leads to better estimates of the treatment means and allows the variability of the
population to be estimated. With several replicates of a treatment, variability about
the mean can be estimated and the credibility of the mean is established. It is the
variability about the treatment mean that is used to compare treatment performance.
Treatments should be replicated for a minimum of three times. Increasing the num-
ber of replicates improves the credibility of the results but also increases the cost
and time involved in running an experiment. These factors can lead to researchers
unknowingly allowing their experiments to be pseudoreplicated.

1.2.8 Pseudoreplication
The term “pseudoreplication” is used to describe the situation where there is repeated
application of treatments but not “true” replication of treatment for reasons of lack of
independence or fallible randomization (Hurlbert, 1984). This can lead to potentially
incorrect statistical analysis and misinterpretation of the results.
In our survey of published papers, only 22% of papers studied could confidently
be classified as not incorporating pseudoreplication. Pseudoreplication was clearly
identified in 6% of the studied papers. The remaining 72% of papers failed to provide
sufficient detail to determine if pseudoreplication had or had not occurred. This lack
of clarity in descriptions of experimental designs made the validity of conclusions of
each experiment difficult to assess.
The following example demonstrates a common pseudoreplication scenario in
postharvest experiments. The experiment aimed to compare the effect of two storage
temperatures on banana ripening (Figure 1.2). In this case, there are four replicates
of the two storage temperatures. Each treatment was applied to an experimental unit
comprised of a tray of 10 bananas. Measurements were made on individual bananas
and an average of 10 measurements used to represent each replicate. In the statistical
analysis, it is important to correctly identify the experimental unit as 10 bananas, not
an individual fruit.
Ideally, we would have enough cool rooms to supply true replicates of the tem-
perature treatments. In this case, four replicates of the two temperatures require
eight different cool rooms (Figure 1.2a). This is often impractical and researchers
may resort to a pseudoreplicated experimental design, where the different replicates
are located within the same treatment cool room (Figure 1.2b). Whilst we recognize
this is the day-to-day reality for many researchers, the temptation to draw inferences
as if the treatments were properly replicated should be avoided or at the very least
acknowledged.
Arguments against conducting pseudoreplication usually address three main
areas of statistical concern: (1) variation, (2) independence of experimental units,
and (3) undetected events.

1.
Variation: Treatments are usually assessed by comparing their observed
impact on the variability inherent in the experimental material. The pur-
pose of replication is to allow a measure of that variability to be made.
Postharvest Technology Experimentation 9

(a)

Rep 2 Rep 3 Rep 4

Rep 1

Temperature 1 Temperature 2 Temperature 2 Temperature 1

Rep 1 Rep 2 Rep 3 Rep 4

Temperature 2 Temperature 1 Temperature 1 Temperature 2

(b)

Rep 1 Rep 2
Rep 1 Rep 2

Rep 3 Rep 4
Rep 3 Rep 4

Temperature 1 Temperature 2
(c)

Rep 1 Rep 2 Rep 3 Rep 4

Temperature 1 Temperature 2 Temperature 2 Temperature 1

Rep 1 Rep 2 Rep 3 Rep 4

Temperature 2 Temperature 1 Temperature 1 Temperature 2

Time 1 Time 2 Time 3 Time 4

FIGURE 1.2  (a) Representation of bananas inside eight different cool rooms set at two dif-
ferent temperatures. (b) Representation of bananas (four hands/“replicates”) inside two cool
rooms set at two different temperatures. (c) Representation of banana storage experiment
replicated at four different times in two different cool rooms at each time.
10 Advances in Postharvest Fruit and Vegetable Technology

A pseudoreplicated experiment will tend to underestimate the random vari-

ation that may occur when the same treatment is applied at another time and
place. In the banana storage example, this occurs when the method of treat-
ment application (in the single room—Figure 1.2b) produces pseudorepli-
cates—the four “replicates” inside each room have actually received the
same treatment. When pseudoreplication occurs, the variability of a single
treatment is confounded with the variability inherent for the single room
in which it is contained. There is no estimate of the background variability
that relates to the process of supplying the temperature that is independent
of the specific chamber in which it is located. Replicate rooms are needed
for each treatment (Figure 1.2a) to distinguish the between-room variabil-
ity from the between-“bananas within trays”-within-room variability (Riley
and Edwards, 1998).
  Apart from the difference in temperature between the different cool
rooms, there are many other characteristics such as lighting, temperature
gradients, and the presence of ethylene and volatile organics that may
vary from room to room, even though attempts are made to control these
features.
2.
Independence of experimental units or true replicates: Another argu-
ment against pseudoreplication involves the concept of independence of
the experimental units. In the banana temperature experiment, when the
bananas were placed in the two different cool rooms, there may have been a
lack of independence present, and consideration must be given to the extent
to which one fruit’s response influences the responses of the other fruit. For
example, one banana in a bunch may have an unnoticed bruise, which may
initiate wound-induced ethylene production. This may induce general rip-
ening of the whole hand (depending on maturity) and could extend to other
replicates within the cool room.
  Lack of independence can also be seen in controlled-atmosphere storage
experiments where a gas for a treatment is supplied from a single source.
Each replicate of a treatment should ideally have its own independent
management system. In that way a mechanical failure, technical failure,
or contamination event will only affect a single experimental unit and be
unlikely to produce a “treatment effect” (Hurlbert, 1984). Although this
may be impractical, these limitations should be recognized and the conclu-
sions interpreted in the light of possible pseudoreplication.
3.
Undetected events: When a treatment is prepared only once, and this sin-
gular source is then supplied to all the different “replicates,” an undetected
problem with the treatment application is more likely to affect all the
repeats. An observed treatment effect may be biased, either positively or
negatively, without the experimenter’s knowledge. For example, if a post-
harvest dip solution is inadvertently made up incorrectly, then all the differ-
ent replicates are treated from this one incorrectly prepared solution.

Arguments in favor of accepting pseudoreplication in postharvest experiments

usually include logistic and cost considerations. For example, it may be impractical
Postharvest Technology Experimentation 11

to have multiple cool rooms set at the same temperature, since resources are limited.
It can be argued that experimental methods used are so well monitored with data
loggers that any unusual events will be recorded and not attributed to particular
treatments. The technical expertise possessed by postharvest researchers is also put
forward as a justification for the practice of pseudoreplication. Although mistakes in
treatment application are rare, they can happen.
In many areas of science, similar treatments are often included in experiments
conducted by other researchers in different places and times. The pseudoreplicated
experiment can then become a “true” replicate in a broader body of knowledge using
meta-analysis (Cottenie and De Meester, 2003; Hannah, 1999).
Pseudoreplication should be avoided in postharvest experiments, although, when
it is present in a study, there are different ways to deal with it: (1) acknowledgment,
(2) limit the inference, (3) replicate in time, and/or (4) use of regression models in
the analysis.

1.
Acknowledgment: Some people acknowledge that they have pseudorep-
licated their experiment but still analyze their data as if these were true
replicates. Osunkoya and Ash (1991) reported: “As the shade house com-
partments were not replicated, differences among treatments may be con-
founded with other sources of error. In the statistical analysis of the data,
it is assumed that the differences in seedling responses among compart-
ments are due solely to the differences in light regime.” This acknowledg-
ment allows the reader to assess these assumptions and consider the paper
accordingly.
2.
Limit the inference: In some pseudoreplicated experiments, other authors
clearly limit the inference drawn from the results. Shah et al. (2000) included
the following statement: “ANOVA could not be performed to directly com-
pare the effects of either different drying humidities or storage conditions.
Such analyses would have been statistically inappropriate and examples of
pseudoreplication due to isolative segregation. The present work should,
however, be seen as indicative of drying and storage conditions which
should be further investigated to obtain more robust guidelines.”
  If the scope of inference of an experiment is limited to the particular time
and place in which the experiment was conducted, then the experiment is
more accurately described as a pilot study. Pilot studies are often under-
taken to assess possible promising treatments for further research, so true
replication is not as important. The scope of inference is confined to the
time, place, and individuals used in the pilot study (Phillips, 2002). “It is an
acceptable analysis if it is recognised that the results are only relevant to
the individual or group of individuals used for the study” (Phillips, 2002).
3.
Replicate in time: If resource availability does not permit true replica-
tion of treatments, repeating the experiments in time can resolve this
issue. Treatment allocations are rerandomized at each time (Figure 1.2c).
Conclusions will be more robust with, say, a different batch of fruit, or fruit
from a different season, and the scope of inference will be wider. However, a
constant issue in postharvest science is seasonality of horticultural produce.
12 Advances in Postharvest Fruit and Vegetable Technology

Generally, there is only a narrow window during which harvesting occurs

and experiments can be conducted. Even if the experiment is repeated at
different times in the same growing season, the same variety may not be
available and the conditions under which the produce has been grown are
likely to be different. Each of these factors may affect storage behavior and
are often given as the reason for pseudoreplication. On the other hand, if a
treatment effect persists across a season and/or between varieties, the treat-
ment effect would be more robust.
4.
Regression models: Statistical analysis methods, such as linear regression,
are suitable to use when treatments are quantitative levels (e.g., storage
responses at 0°C, 2°C, 5°C, 10°C, and 15°C) and there has been no rep-
lication. Linear mixed effects regression models, which allow for correla-
tion among residuals, may also be used to analyze pseudoreplicated data
(Pinheiro and Bates, 2000).

1.2.9 Sub-Sampling
It is common that journal articles report experiments that were conducted “in trip-
licate.” A reader is left to wonder whether there were three true replicates of each
treatment, or whether a single batch of experimental material was prepared for each
treatment and then divided into three sub-samples for analysis (triplicates). The dis-
tinction between the two scenarios is critical for the correct data analysis and inter-
pretation of results. An example is the extraction and measurement of anthocyanins,
where an experimental unit comprised of five apples that were blended into a liquid,
from which three sub-samples were taken and measured. This is a triplicate sub-
sample from one replicate, and measures the variability of the analytical method.
Data from each sub-sample triplicate is not independent and needs to be averaged to
obtain one value to represent the experimental unit (five apples). Unfortunately this
is an extremely common error. To validly compare the effect of treatments, indepen-
dent replications are necessary. For example, for each treatment, extractions from
three different replicates of five apples (where each replicate has been treated inde-
pendently) are required. Pooling of variation is further discussed in Section 16.2.4.

1.2.10 Incorrect Statistical Analysis

The most appropriate type of statistical analysis to use is determined by the experi-
mental design and the type of data collected in the study. Some recommended texts
to assist experimental design and statistical analysis include Steel and Torrie (1980),
Ramsey and Schafer (2002), Moore et al. (2012), and Welham et al. (2014).
Data arising from a single experiment is just one realization of the many, many
experiments that could have occurred under similar conditions. Statistical analy-
sis allows statements to be made about the population of experiments of which
the observed data is only a sample (Snedecor and Cochran, 1967). The data from
one single experiment provides information about the particular system that we are
studying and it is the system that is the focus of modeling, not the data. A discussion
Postharvest Technology Experimentation 13

and summary of current techniques in postharvest modeling are p­resented in

Chapter 16.
In postharvest experiments, there are generally four types of data collected: (1)
binomial (e.g., yes/no), (2) ordinal (e.g., quality scores), (3) discrete (e.g., count of
rotten fruit), and (4) continuous (e.g., weight in grams). It is important to identify the
data type as each requires a specialized statistical analysis method and analysis of
variance (ANOVA) is not always the most appropriate data analysis method to use.
Methods for statistical analysis of each data type are described below.
1. Binomial data: Binomial data has traditionally been transformed using the
arcsine (or angular) transformation of proportions prior to ANOVA. However, better
methods such as generalized linear regression models (GLMs) can be used to ana-
lyze this type of data. Residuals follow the binomial distribution and a logistic (logit)
link function is commonly used. Instead of analyzing the actual proportion, a GLM
with a logistic link function analyzes the logarithm of the odds of the event occuring.
All the statistical analysis and treatment comparisons are made on the logistic scale
and can be back transformed to proportions for presentation. The probit link function
is also available for binomial data.
2. Ordinal data: Subjective assessment of fruit and vegetables using scoring tech-
niques is common in many postharvest experiments. This data type has a defined
scale, often on a 1–5 subjective scale. This data is not continuous and ANOVA
assumptions are unlikely to be met. However, when an experimental unit comprises
multiple single fruit and, having several different people independently assess each
fruit, the average of all the scores for each unit can be assumed continuous and ana-
lyzed using ANOVA after carefully checking that the residuals assumptions are met.
Ordinal linear regression methods are also available to analyze score data.
3. Discrete (count) data: While ANOVA of log-transformed count data is com-
mon, a GLM with Poisson error distribution and log link function is recommended.
When counts are converted to percentage data, it is preferable to analyze the raw
count data using a GLM with binomial errors.
4. Continuous data: Linear models ANOVA has been a standard method for
comparing treatments from designed experiments with continuous data. ANOVA
is a type of linear model widely used to test treatment effects when data originates
from a continuous scale of measurement (e.g., fruit weight). ANOVA is a method
for comparing means and the term “variance” should not be misleading. The word
“variance” relates to the approach to assess differences in treatment means by com-
paring amounts of variability explained by different sources. The overall variability
is calculated by squaring differences between each data value and the overall mean,
and then adding each squared difference together.
After using ANOVA to determine differences between treatment means, the
unexplained variability (residuals) requires consideration. There are three assump-
tions, related to residuals from the model, need to be met for a valid ANOVA.
These are

1. Independence (residuals from one experimental unit are not influenced by

residuals from any other unit).
14 Advances in Postharvest Fruit and Vegetable Technology

2. Identical (residuals have a constant variance regardless of the size of the

fitted value).
3. Normally distributed (a histogram of residuals resembles the bell shape of
the normal distribution).

All of these assumptions need to be tested and met to conduct a valid ANOVA. A
correct analysis of the data ensures that sound conclusions can be drawn from the data.

Permutation test: The permutation test is an alternate method for testing treat-
ment effects when either the normality assumption is not met, there are outliers, or
the sample size is too small. Although this test is not commonly used in postharvest
statistical analysis, it could be used with data that does not meet the assumptions
of ANOVA (Welham et al., 2014). This approach has been applied in postharvest
science, where Bobelyn et al. (2010) examined apple storage behavior, which was
predicted by near infra red (NIR) analyses.
Mixed effects models: Linear modeling is a subset of a larger group of models
known as mixed-effects models (Pinheiro and Bates, 2000). Mixed-effects models
include both fixed and random effects and provide a way to investigate complex pat-
terns of variability that are representative of real world variability (Zuur et al., 2009).
A fixed effect (or factor) is one that the research question identifies (e.g., 1-MCP rate),
while random effects are usually ones that are assumed to represent a broader popu-
lation (e.g., physical blocks). Random factors can also take into account the lack of
independence in residuals, for example, from a repeated-measures experiment where
the data comprises responses from exactly the same apple taken on several occa-
sions over a period of time. Mixed-effect models also allow the analysis of data from
unbalanced experimental designs where there are unequal numbers of replicates of
each treatment.

1.2.11 Reporting Statistical Analysis Methods and Results

In our survey of published postharvest papers, we found that 62% of the papers had
either no or poor statistical methods descriptions, while 56% had poor presentation
of the results. Common failings in the reporting of statistical analysis methods and
results include:

1. Not detailing the actual treatment factors being tested and experimental
design structure used; the reader needs to have confidence that the factors
were correctly structured and tested in the analysis. An incorrect design
structure in the model will lead to incorrect conclusions.
2. Not identifying measures of variability—for example, stating whether
the variability is a standard deviation, standard error, or least-significant
difference.
3. Not using appropriate wording to describe the statistical analysis—for
example, saying that “data were analyzed using Statistica” is not sufficient.
In addition, terms encountered in published papers, such as “complete ran-
domized block,” are incorrect.
Postharvest Technology Experimentation 15

4. Exaggerating the precision of results by using too many decimal places—for

example, the average SSC has been reported as 13.256 Brix. This measure-
ment should be reported as 13.3, since the precision of most refractometers
is in increments of 0.1.

Statistical significance was historically indicated using as asterisk system where *

represented p < 0.05, etc. However when reporting on the results, it is more useful
to supply the actual test statistic (e.g., F value), appropriate degrees of freedom and
actual probability value for all treatment effects where appropriate. For example, in
a trial of avocado storage, where the results were critical to industry, the treatment
effect and treatment means could be reported, even though the calculated F value
was not significant (F3,6 = 0.69, P = 0.593). When actual probability values are sup-
plied, a single experiment is more easily incorporated into any future meta-analysis,
thus increasing the value of the research (Fidler, 2010). Indeed, some online journals
provide the opportunity to include supplementary data that support the manuscript.
The supply of this additional data not only allows the reader to scrutinize the data to
confirm results and conclusions, it also allows the potential for future meta-analysis
and more open research.
Another approach to data-reporting is presenting effect sizes, by supplying treat-
ment means within the text and providing confidence intervals (usually 95%) for
means, rather than just stating that an effect was significant (Cummings, 2012). The
confidence interval for the mean provides a range that is highly likely (often 95% or
99%) to contain the true population mean.

1.2.12 Statistical Significance versus Biological Significance of Results

On some occasions, the differences between postharvest treatments that have been
declared significant according to a statistical test do not always relate to biological or
commercial differences. For example, there may be a statistically significant treat-
ment effect on peel color (as measured by the Minolta color meter), but the actual dif-
ference in color between the treatments may not be detectable to the eye or have any
commercial significance. It is important to relate the statistically analyzed results to
biological and commercial significance.
After conducting a statistical analysis, researchers commonly investigate differ-
ences between treatments and want to know if an observed effect is “statistically
significant.” The statistician R.A. Fisher recommended a probability level (P value)
of 1 in 20 (or 5%) in his classic book Statistics Methods for Research Workers as
being “convenient to take this point as a limit in judging whether a deviation is
considered significant or not. Deviations exceeding twice the standard deviation are
thus formally regarded as significant” (Fisher, 1938). This convention has since been
widely adopted in science.
P is the probability (assuming there is no difference between treatment means)
of obtaining a test statistic (e.g., F value) as large, or even larger, than the one that
was observed. According to the Fisher convention, a treatment effect with a P value
greater than 0.05 would be reported as being “not significant.” However, it may be
appropriate to report that there was evidence of a treatment effect and, by including
16 Advances in Postharvest Fruit and Vegetable Technology

the actual P value, a reader can interpret the treatment effect in the context of the
replicated and designed experiment, for example, when P = 0.051.
When a test statistic is not significant (usually P > 0.05), instead of writing “there
was no difference between the treatments,” it is more appropriate to write “no sig-
nificant difference was detected between the treatments.” In a small experiment,
say, with only three replicates of four treatments, there will sometimes be a “non-
significant” treatment effect that is real and important. However, if the experiment
had been larger, say, six replicates of the same four treatments, this effect may have
been detected as being statistically significant.
Although using P values provides a convenient guide to help delineate statistical
significance, other knowledge about the system being studied and the context of the
experiment is also important. Cummings (2012) claims that P values are unreliable
and, instead, suggests that confidence intervals provide a better estimate of the true
treatment effect, and also indicate the extent of uncertainty in the result.
People also sometimes misinterpret the P value as the probability that the treat-
ment being studied actually works.

1.3 CONCLUSIONS
A sound postharvest experiment manages variability and clearly articulates the popu-
lation to which the conclusions will apply. It provides sufficient details of the experi-
mental design, protocols, statistical analysis, and results. When all of these factors are
all appropriately addressed, conclusions based on the results from the experiment will
be robust and valid. Subsequent postharvest treatment recommendations can then be
assessed with confidence by industry, thus potentially improving the availability and
quality of horticultural produce.

ACKNOWLEDGMENT
The authors gratefully acknowledge the contributions of Beverley Orchard and
Sharon Nielsen as codevelopers of the NSW Department of Primary Industries
Biometrics Basic Statistics course where many of the ideas expressed in this chapter
originated. They also acknowledge the critical reading of this chapter by Daniel
Valero and Chris Watkins.

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 2 Recent Research
on Calcium and
Postharvest Behavior
Babak Madani and Charles F. Forney

CONTENTS
2.1 Introduction..................................................................................................... 19
2.2 Postharvest Diseases........................................................................................20
2.3 Physiological Disorders................................................................................... 21
2.4 Quality and Ripening of Climacteric Fruit..................................................... 23
2.5 Quality of Nonclimacteric Fruit...................................................................... 26
2.6 Fresh Cuts........................................................................................................ 27
2.7 Conclusions...................................................................................................... 28
References................................................................................................................. 29

2.1 INTRODUCTION
Calcium is essential for the growth and development of plants. Calcium moves in
soil mostly by mass-flow, and its uptake by plants is passive and restricted to the tips
of young roots with nonsuberized endodermal cell walls. Calcium is translocated in
the xylem mostly through the transpiration stream. Calcium moves upward in the
xylem by adsorption onto exchange sites and by chelation with organic acids in the
xylem sap. High concentration of calcium in the xylem sap facilitates its movement
to the shoot apex (Biddulph et al., 1961; Bell and Biddulph, 1963). Preharvest appli-
cation of calcium can be applied to the soil or foliage. Application of calcium in the
soil will not always increase the concentration of calcium in fruits or vegetables,
because of the immobility of calcium inside the plant and competition for calcium
among different plant parts. Also, calcium can take up to four years to move from
the roots to the leaves or fruits (Jones et al., 1983; Yuen, 1994). Calcium can also
be applied as a foliar spray or by postharvest application to the produce. When
calcium is applied to fruit, it enters through trichomes, stomata, and lenticels (Scott
and Wills, 1975; Glenn et al., 1985). Calcium uptake into leaves also depends on
stomata channels. Transportation of calcium to nonvascular flesh tissue is by dif-
fusion through the apoplast. Stomata density also can influence calcium uptake by
fruits or leaves (Harker et al., 1989). In the European Union, there are many com-
mercial foliar fertilizers containing various forms of calcium, but their efficiency

19
20 Advances in Postharvest Fruit and Vegetable Technology

of uptake as a preharvest spray is seldom more efficient than calcium chloride or

nitrate (Wójcik and Szwonek, 2002).
Postharvest application of calcium to fruits and vegetables is generally accom-
plished by dipping in solutions that primarily utilize calcium chloride, but lactate,
propionate, gluconate, and nitrate salts have also been reported to be effective.
Vacuum infiltration enhances the penetration of calcium solutions into cells through
the pressure generated after release of the partial vacuum and has been shown to be
very effective in modifying postharvest changes in a range of produce (Scott and
Wills, 1975, 1977a,b, 1979; Wills and Scott, 1980; Tirmazi and Wills, 1981; Wills
and Tirmazi, 1982; Wills et al., 1982, 1988a,b; Wills and Sirivatanapa, 1988; Martin-
Diana et al., 2007).

2.2  POSTHARVEST DISEASES

Economic losses caused by postharvest pathogens have resulted in the development
of a range of cultural, physical, and chemical treatments to control postharvest dis-
eases. The application of synthetic fungicides has been a major treatment for control-
ling many diseases, but use of any compound for an extended time invariably leads
to an increase in fungicide-resistant fungal strains. In addition, potential fungicide
residues on fruit can be an issue for consumers. These concerns have encouraged the
development of safer approaches to disease management.
The application of calcium is an alternative to the use of fungicides. Calcium has
been shown to reduce disease in crops by inhibiting spore germination and stimulat-
ing host resistance. Calcium also acts by stabilizing cell walls to make them more
resistant to harmful enzymes released by fungi (Sams and Conway, 1984; Wisniewski
et al., 1995; Biggs, 1999). In addition, the calcium ion inhibits the action of ethylene
on cell membranes and thereby inhibits the onset of senescence (Torre et al., 1999).
High concentrations of cytosolic calcium have been shown to enhance the synthesis
of phytoalexins and phenolic compounds that decrease the activity of pathogenic
pectolytic enzymes (Miceli et al., 1999).
In this section, we focus on the effect of calcium on anthracnose (Colletotrichum
spp.), brown rot (Monilinia fructicola (G. Wint.) Honey), gray mold (Botrytis cine-
rea), green mold (Penicillium digitatum), and sour rot (Geotrichum citriauranti).
Anthracnose is a postharvest disease that affects a range of fruits, including
papaya, banana, dragonfruit, and strawberry (Chau and Alvarez, 1983; Kim et al.,
1992; Ghani et al., 2011). In papaya, the effect of pre- and postharvest calcium has
been studied by Madani et al. (2014a) who reported that 1.5% and 2% calcium chlo-
ride significantly reduced anthracnose incidence and severity during five weeks of
storage while Mahmud et al. (2008) found that the lowest incidence of anthracnose
was obtained in fruit infiltrated with 2.5% calcium chloride. Awang et  al. (2011)
further reported that postharvest dipping of dragonfruit in 1–4 g/L calcium chloride
did not have significant effect on the incidence of anthracnose but the size of lesions
was reduced with increasing calcium concentrations. On the other hand, Ghani et al.
(2011) indicated that preharvest 1%–4% calcium chloride dips reduced the severity
of anthracnose disease in dragonfruit. Chillet et al. (2000) showed that the suscepti-
bility of the banana fruit to anthracnose was lower in fruit with higher calcium levels.
Recent Research on Calcium and Postharvest Behavior 21

However, Nam et al. (2006) did not find any effect of 1–6 mM calcium in the nutrient
solution used for growing strawberry plants on the severity of anthracnose.
Brown rot affects most commercially grown Prunus spp. and can result in exten-
sive crop losses (Adaskaveg et al., 2008). Early studies showed that the application
of calcium chloride did not reduce the incidence of postharvest brown rot in peaches
(Conway, 1987). However the use of calcium chloride as a foliar spray applied up to
six times was shown to increase fruit Ca concentration and reduce the occurrence
of brown rot (Manganaris et al., 2005; Elmer et al., 2007). In New Zealand, foliar
sprays of calcium have been widely adopted by stonefruit growers as a practical tool
to reduce brown rot. Biggs et al. (1997) tested the effects of several calcium salts
on the in vitro growth of Monilinia fructicola and found that calcium propionate
strongly inhibited fungal growth.
Gray mold rots can cause severe postharvest losses in grapes and strawberry fruit
(Bulger et al., 1987; Wilcox and Seem, 1994; de Kock and Holz, 1994; Hernandez-
Munoz et al., 2006). Spraying calcium chloride 2–3 times before veraison, increased
the Ca concentration of grape berries and reduced rot caused by gray mold (Amiri
et al., 2009; Ciccarese et al., 2013). Singh et al. (2007) also showed that spraying cal-
cium on strawberry could decrease gray mold. The lower incidence of rot might be
due to calcium slowing the senescence processes in many fruit, including strawberry
(Ferguson, 1984; Poovaiah, 1986; Sharma et al., 2006). Wójcik and Lewandowski
(2003) and Naradisorn et al. (2006) have also reported that the fruit receiving calcium
is much firmer and less affected by gray mold. Similarly, Lara et al. (2004) reported a
lower incidence of gray mold in strawberry after postharvest calcium dipping.
Green mold (Penicillium digitatum) and sour rot (Geotrichum citriauranti) are
the most economically important postharvest diseases of citrus in the arid fruit-
growing regions of the world (Powell, 1908; Eckert and Brown, 1986). Smilanick
and Sorenson (2001) indicated that incidence of green mold was greatly reduced
by dipping lemons or oranges in a 40°C liquid lime–sulfur solution that contained
0.75% (w/v) calcium polysulfide. The incidence of sour rot was also reduced by
this treatment. Efficiency was higher on lemons than oranges, and on green- com-
pared to yellow-lemons. Youssef et al. (2012) reported that the efficiency of pre-
harvest sprays of calcium chloride and calcium chelate against postharvest green
and blue mold on “Comune” clementine and “Valencia late” orange fruit were
greater than using a postharvest dip. They concluded that field application of cal-
cium should be included in an integrated approach for controlling postharvest
diseases of citrus fruit.

2.3  PHYSIOLOGICAL DISORDERS

Physiological disorders involve the breakdown of plant tissue that is not directly caused
by pests and diseases or by mechanical damage. They may develop in response to var-
ious pre- and postharvest conditions, including nutrient accumulation during organ
development or low temperature stresses during storage (Wills et al., 2007). Several
disorders have been associated with calcium deficiency, which include blossom end
rot (BER) in tomato, glassiness in melon, bitter pit in apple, cracking in cherry, and
chilling injury of various commodities in storage (Wills et al., 2007).
22 Advances in Postharvest Fruit and Vegetable Technology

Bitter pit (BP) is a major problem for an apple industry, especially where grow-
ing conditions are dry. Application of calcium either pre- or postharvest has been
known for many years to reduce the susceptibility of apples to develop BP (Shear,
1975; Wills et al., 1976; Scott and Wills, 1977b, 1979; Ferguson and Watkins, 1989;
Conway et al., 1991, 1994; Fallahi et al., 1997; Ferguson et al., 1999; Blanco et al.,
2010). In some recent studies, Benavides et  al. (2001) showed that the greatest
increase in calcium concentration in apples was achieved when calcium was sprayed
six times at 15-day intervals, from 60 days after full bloom, while Lötze and Theron
(2007) showed that application of foliar calcium during midseason (40 days after full
bloom) was shown to be more effective in increasing fruit calcium and reducing BP
than a later application. Neilsen et al. (2005) showed that sprays of calcium chloride
in the early growing season were as effective as in the late season for reducing BP, in
spite of low calcium concentration in the harvested fruit. The reason for this effect is
unclear. Blanco et al. (2010) showed that spraying the apple trees with calcium chlo-
ride or calcium propionate in combination with carboxymethyl ether of cellulose can
favor the distribution of calcium into the apple fruit and help to reduce the incidence
of calcium-related disorders during cold storage.
Internal browning (IB) is a low-temperature disorder affecting a number of fruits
with the browning of the flesh tissue occurring after removal from cool storage to
room temperature (Teisson et  al., 1979; Smith, 1983; Paull and Rohrbach, 1985).
Youryon et al. (2013) mentioned that calcium infiltration of pineapple via the pedun-
cle for three days increased calcium concentrations in the core and adjacent flesh
tissue and reduced IB in “Trad-Srithong” pineapples stored at 13°C. Wójcik (2012)
showed that six sprays of calcium chloride on pear fruit—from six weeks after full
bloom to two weeks before harvest—resulted in firmer and greener fruits that were
less sensitive to IB after storage. Manganaris et al. (2007) found that dipping peach
in calcium chloride for 5 min decreased flesh browning after storage at 5°C.
Cracking in sweet cherries and litchi, caused by rainfall shortly before harvest,
limits the production of sweet cherry in many parts of the world (Sekse, 1998; Huang,
2005). Many studies have examined the potential to reduce cracking in cherry fruit
by spray-application of calcium compounds such as calcium chloride, hydroxide, and
nitrate (Callan, 1986; Meheriuk et al., 1991; Yamamoto et al., 1992; Rupert et al.,
1997; Marshall and Weaver, 1999). Erogul (2014) sprayed sweet cherry trees with
calcium nitrate, chloride, caseinate and hydroxide 30, 20, and 10 days before har-
vest, and showed the most effective applications to decrease cracking were calcium
hydroxide and calcium chloride. However, Huang et al. (2008) found that spraying
with calcium chloride three times did not decrease cracking in litchi.
It is widely accepted that BER of tomato is caused by a combination or sequence
of environmental and physiological factors related to calcium uptake and transport
within the plant, which affect the fruit’s growth rate and/or size (Ho and White,
2005). The majority of studies identify a local calcium deficiency in distal fruit tis-
sue during the period of rapid cell expansion (7–21 days after anthesis), when cal-
cium demand exceeds supply, as the primary cause of BER (Bradfield and Guttridge,
1984; Adams and Ho, 1993; Ho and White, 2005). Additional application of cal-
cium is commonly considered as a preventive measure to overcome local calcium
deficiency in tomato (Wada et al., 1996; Ho, 1999; Schmitz-Eiberger et al., 2002).
Recent Research on Calcium and Postharvest Behavior 23

Liebisch et al. (2009) showed that spraying tomato plants with calcium chloride and
boric acid decreased BER, but increased cracking in the fruit. They concluded that
since sprays are costly, labor-intensive, and did not reduce the proportion of nonmar-
ketable fruit, the selection of cultivars, not susceptible to BER and cracking is more
effective than sprays, when conditions favor these disorders. This is particularly true
for protected cultivation in central Thailand. However, Saure (2014) suggested that
the actual causes of BER are the effects of abiotic stress (such as salinity, drought,
high-light intensity, heat, and ammonia nutrition) resulting in an increase of reac-
tive oxygen species, high oxidative stress, and, finally, cell death. Cell death results
in a disintegration of the plasma membrane and tonoplast and a breakdown of the
endoplasmic reticulum, thus not following but preceding ion leakage, including Ca2+
leakage, and loss of turgor. With this approach, a better understanding and a more
efficient control of BER in tomato and pepper fruit is envisaged.
Glassiness is a disorder in which the melon flesh appears water-soaked; this has
been related to low calcium concentration in fruit tissue (Lester and Grusak, 1999,
2001; Madrid et  al., 2004). Serrano et  al. (2002) confirmed that this disorder is
related to calcium deficiency by decreasing calcium in the nutrient solution, which
caused a more rapid onset of softening of the fruit and increasing glassiness.

2.4  QUALITY AND RIPENING OF CLIMACTERIC FRUIT

In climacteric fruit, ripening is accompanied by a rise in respiration rate and ethyl-
ene production (Wills et al., 2007). It has been proposed that calcium delays ethyl-
ene production by preventing solubilization of calcium binding sites in cell walls,
which activates the ethylene generation system located in the cell-wall plasma mem-
brane complex (Mattoo and Lieberman, 1997). Wang et  al. (2006) found that the
expression of LeACO1 gene (related to changing ACC to ethylene) was inhibited
with calcium application in never-ripe mutant and wild-type tomatoes at the green
stage. Calcium also decreases the respiration rate in climacteric fruit by regulation
of membrane fluxes of initiator molecules like phosphate or respiratory substrates
like malate (Ferguson, 1984). Phosphate flux changes across the tonoplast and plas-
malemma have been associated with an onset of the climacteric respiratory rise.
It is suggested that the increased tonoplast and plasmalemma fluxes of phosphate
and increased cytoplasmic phosphate may be critical for climacteric respiration
(Woodow and Rowan, 1979). The tonoplast and plasmalemma could be the pos-
sible site for calcium to regulate phosphate. Other substrates such as malate, which,
through decarboxylation, may be a source for CO2 in the climacteric phase, may be
regulated by calcium in terms of compartmentation between vacuole and cytoplasm
and, thus, decrease the respiration rate (Ferguson, 1984).
This section will discuss recent findings on the role of calcium in the postharvest
quality of selected pome fruits (apple and pear), stone fruits (plum and peach), tropi-
cal fruit (papaya), small fruit (blueberry), and tomato and melon.
In apples, the effects of pre- and postharvest calcium in postharvest quality
were evaluated in many early studies (Wills, 1972; Mason, 1976; Scott and Wills,
1977a; Tirmazi and Wills, 1981; Sams and Conway, 1984). More recently, Kadir
(2005) showed that spraying apple trees six times with calcium chloride improved
24 Advances in Postharvest Fruit and Vegetable Technology

“Jonathan” apple quality in terms of fruit weight, size, appearance, and color, but
had no effect on soluble solids content. Similar findings were noted by Wójcik and
Borowik (2013), who found that apples sprayed with a range of calcium compounds
had higher titratable acidity and were firmer, but did not differ in soluble solids. Val
et al. (2008) found that 1% calcium chloride sprays had no effect on the concentra-
tion of calcium in the flesh of “Fuji” apples and no effect on fruit quality traits; Wu
et al. (2012) showed that postharvest calcium dipping of “Fuji” with 0.5% calcium
chloride was effective in maintaining the firmness of cut-apple products and intact
fruit. Similarly, Zheng et  al. (2014) reported a 2% calcium chloride dip on intact
“Fuji” maintained firmness and improved surface color and titratable acidity of cut
apple fruit. Hussain et al. (2012) showed that “Red Delicious” apple fruit dipped with
2% calcium chloride and then irradiated with 0.4 kGy gamma radiation had higher
ascorbic acid levels and extended shelf-life. Ortiz et al. (2010) showed that most of
the compounds that contribute to flavor in ripe apple were improved in response to
a postharvest calcium chloride dip. For example, they showed that the release of
acetate esters was favored, and acetaldehyde content was enhanced, in apple fruit
treated with calcium (Ortiz et al., 2010). They further suggested that this technique
may be an appropriate method to improve the fruit’s aroma where these effects were
from an increase in pyruvate decarboxylase and alcohol dehydrogenase activities in
the presence of calcium. Ortiz et al. (2011) indicated that preharvest calcium sprays
enhanced most of the compounds contributing to overall flavor in ripe fruit, suggest-
ing that this procedure may be suitable for improving the fruit’s aroma at harvest.
The positive effects of calcium in enhancing postharvest quality of pear have
confirmed earlier studies by Wills et al. (1982), Raese and Drake (1993, 1995) and
Raese et al. (1999). Wójcik et al. (2014) examined the impacts of autumn sprays of
calcium as a supplement to summer-time calcium sprays on “Conference” pear qual-
ity and storability. They found that the greatest increase in calcium status was in
fruit treated with calcium in the summer and in the fall. After storage, pears sprayed
with calcium in the summer and in the fall produced less ethylene, had lower res-
piration, contained more organic acids and were firmer (Wójcik et al., 2014). They
concluded that calcium chloride at 20 or 25 kg/ha should be used in “Conference”
pear orchards as a supplement to summer-time calcium sprays to improve fruit stor-
ability. Mahajan and Dhatt (2004) studied the effects of different dip concentrations
of calcium chloride and showed that application of 4% calcium chloride was the most
effective treatment in reducing the weight-loss of pear and in maintaining the firm-
ness and quality of fruits up to 75 days in storage at 0–1°C.
In plum, the beneficial effects of calcium treatment—in terms of prolonging
storability, delaying the ripening process and maintaining fruit quality—has been
reported by Valero et al. (2002). Serrano et al. (2004) studied the role of posthar-
vest treatments with calcium or heat on reducing mechanical damage during stor-
age. They showed that 1 mM calcium chloride dips or hot-water dips at 45°C for
10 min led to a reduction of mechanical damage, and in turn alleviated the physi-
ological responses that occurred in mechanically damaged plums. Alcaraz-Lopez
et al. (2003) studied the combined effects of foliar applications of Ca, Mg, and Ti on
the calcium nutrition and fruit quality of plum and found that titanium application
increased the calcium concentrations in the fruit’s peel and flesh.
Recent Research on Calcium and Postharvest Behavior 25

Recent studies on calcium in peach have confirmed earlier reports by Abdalla and
Childers (1973) and Wills and Mahendra (1990). Research on peach in the eastern
USA showed that calcium sprays were effective, while, in western USA, these treat-
ments did not increase the fruit’s calcium content. Thus, growing conditions and
cultivar determine calcium absorption into fruit (Johnson et al., 1998; Crisosto et al.,
2000). Manganaris et al. (2005) found that preharvest calcium sprays increased cal-
cium in peach, but did not change acidity, soluble solids, and ethylene production
after harvest. Manganaris et al. (2007) found that peach dipped in calcium lactate,
propionate, and chloride solutions, maintained firmness during storage. Gupta et al.
(2011) showed that peach dipped in calcium chloride had reduced weight-loss and
maintained firmness, acidity, and vitamin A content during storage.
Mahmud et al. (2008) studied the effect of dipping or infiltration with calcium
chloride on the postharvest quality of papaya and found that vacuum infiltration was
more effective than dipping at ambient pressure for maintaining quality of papaya
“cv. Eksotika II.” However, Qiu et al. (1995) demonstrated that spraying or dipping
papaya (“cv. Kapoho Solo”) in calcium chloride did not increase calcium concen-
tration in the fruit’s mesocarp. In contrast, Madani et  al. (2014b) concluded that
the foliar sprays of papaya (“cv. Eksotika II”) with calcium chloride resulted in an
increased calcium concentration in its peel and pulp tissues, firmness, and titratable
acidity and overall fruit quality, but reduced respiration rate, ethylene production,
and soluble solids concentrations.
A factor limiting the postharvest life of blueberries is excessive softening
(Lambert, 1990) and this has been shown to be positively affected by dipping in
calcium chloride (Hanson et  al., 1993). Stuckrath et  al. (2008) studied the effect
of foliar application of calcium under three different growing conditions (tunnel,
mesh, and ambient) and found that the fruit’s firmness was higher at the begin-
ning of the cell-expansion period in the tunnel-grown fruit and a linear correlation
between calcium concentration and firmness was established. Angeletti et al. (2010)
evaluated the effect of calcium fertilization of “O’Neal” and “Bluecrop” blueberry
on quality during storage and showed that calcium-treated fruit for both varieties
had less softening and weight-loss compared with fruit without calcium fertiliza-
tion. Angeletti et al. (2010) also showed that although the respiration rate increased
during storage, this was lower in calcium-treated blueberries. They also showed
that the calcium treatments did not alter hemicellulose content, but, in some cases,
reduced solubilization of pectic polymers (Angeletti et al., 2010).
Cut melon is prone to softening during storage, even under modified atmosphere
packaging; calcium has been shown to decrease softening (Madrid et  al., 2004).
Moreover, Saftner et al. (2003) showed that honeydew fruit dipped in calcium pro-
pionate, calcium amino acid chelate formulation, or calcium chloride, decreased
respiration and ethylene production during storage. However, Aguayo et al. (2008)
demonstrated that cut melon dipped in calcium chloride at 60°C maintained firm-
ness during eight days of storage and Luna-Guzmán and Barret (2000) found that cut
cantaloupe dipped in calcium lactate had firmer tissue without bitterness. Johnstone
et  al. (2008) found fertigation with calcium chloride had no effect on quality or
calcium concentration of Californian honeydew or muskmelon, probably due to
the physiological limitation of calcium movement into the fruit tissue. However,
26 Advances in Postharvest Fruit and Vegetable Technology

Lester and Grusak (2004) reported that foliar application of calcium metalosate—or
“Folical”—increased the honeydew fruit’s calcium and storage life, but did not affect
netted cantaloupe.
Early research on calcium and postharvest quality of tomato was reported by
Wills et al. (1977), Wills and Rigney (1979), Wills and Tirmazi (1979), Rigney and
Wills (1981), Minamide and Ho (1993), and Paiva et al. (1998). A recent study by
Senevirathna and Daundasekera (2010) on mature-turning tomato fruit dipped in
calcium chloride at ambient and partial pressure, found vacuum infiltration to be the
most effective treatment with respect to shelf-life extension and reduced ethylene
production. Coolong et  al. (2014) further found that foliar application of calcium
chloride increased fruit soluble solids content and dry weight, but did not affect
texture while weight loss during storage increased. Dong et al. (2004) found that cal-
cium chloride sprayed during anthesis and on one and three-week old fruit improved
vitamin C content and reduced titratable acidity of the fruit.

2.5  QUALITY OF NONCLIMACTERIC FRUIT

The effects of calcium on the postharvest quality of the nonclimateric fruits such as
cherry, pomegranate, grape, and strawberry with emphasis on recent findings will be
discussed in this section.
Studies on the effects of preharvest calcium on quality parameters in cherries
during storage are relatively limited (Lidster et al., 1979). Tsantili et al. (2007) inves-
tigated effects of preharvest sprays of calcium chloride on the quality of “Vogue”
cherries during storage and found calcium-treated fruit had greater firmness, lower
soluble pectin content, and greater resistance to stem removal, but did not affect res-
piration and showed only slight effects on reducing ethylene production. Wang et al.
(2014) futher showed that a postharvest calcium chloride dip treatment reduced the
fruit’s respiration and ethylene production rates.
Ramezanian et al. (2009) examined the effect of calcium on pomegranate quality
and showed that preharvest calcium chloride sprays at full bloom and one month
later increased fruit weight, soluble solids content, and ascorbic acid in the aril.
Kazemi et al. (2013) showed that postharvest dipping in calcium chloride and found
better retention of firmness, vitamin C, and titratable acidity. Ramezanian et  al.
(2010) further showed that pomegranate dipped in calcium chloride or infiltrated
with calcium chloride and spermidine exhibited reduced weight loss and increased
ascorbic acid.
As a consequence of early research on calcium in grapes (Schaller et al., 1992),
calcium chloride is now commonly applied to grapes in Italy and is reported to
decrease decay and delay senescence (Romanazzi et  al., 2012). Ciccarese et  al.
(2013) studied the effect of spraying calcium EDTA before and after veraison and
showed that the fruit maintained berry firmness. The applications were particularly
efficacious if carried out between fruit set and veraison when stomata are functional
and calcium was better adsorbed. Marzouk and Kassem (2011) showed that vines
sprayed with calcium chloride during fruit development until harvest had higher
firmness and improved quality after harvest. Amiri et  al. (2009) examined cal-
cium chloride sprays from fruit set until veraison and showed that grape quality
Recent Research on Calcium and Postharvest Behavior 27

parameters such as juice pH, soluble solids, and titratable acidity were not affected,
whereas berry firmness, berry color, and appearance improved at harvest. However,
contradictory results were shown by Bonomelli and Rafael (2010) who found foliar
and soil application of calcium chloride before veraison had no influence on calcium
and sugar concentration in fruit.
Strawberry shelf-life is limited mostly due to susceptibility to fungus disease.
Singh et  al. (2007) studied effects of preharvest foliar application of calcium and
boron on fruit yield and quality of “Chandler” strawberry. They found no effect
on individual berry weight, but marketable fruit yield increased and was highest
in plants sprayed with calcium plus boron. Calcium-treated fruits were firmer, had
lower soluble solids, and higher acidity and ascorbic acid at harvest (Singh et  al.,
2007). Figueroa et al. (2012) further evaluated the effect of calcium chloride dip-
ping and/or naphthalene acetic acid (NAA) at 45°C and found lower soluble sol-
ids in calcium and NAA treated fruits during cold storage, suggesting that these
compounds could alter the normal breakdown of cell-wall polysaccharides during
postharvest. After storage, calcium chloride in combination with NAA produced a
reduction in the transcriptional level of polygalacturonase, pectate lyase, and EG1
genes. Figueroa et al. (2012) concluded that calcium and auxin could individually
alter fruit ripening, preventing the normal degradation of cell walls during cold
storage, while the combination of calcium and NAA reduced the transcript level of
cell-wall degrading genes after cold storage, albeit no differences in firmness were
recorded. Hernandez-Munoz et al. (2006) studied the effect of postharvest calcium
gluconate and chitosan coatings on quality of strawberry fruit and showed that the
calcium dips were effective in maintaining firmness, while chitosan coatings mark-
edly slowed ripening as shown by their retention of firmness and delayed changes
in their external color. Whilst addition of calcium gluconate to the chitosan coating
formulation did not further extend the shelf-life of the fruit, the amount of calcium
retained by strawberries was greater than that obtained with calcium dips alone
(Hernandez-Munoz et al., 2006).

2.6  FRESH CUTS

In recent years, the demand for fresh-cut products has increased as a consequence
of changes in consumer attitudes (Martin-Diana et al., 2007). However consumers
have the expectation that processing and storage of fresh-cut products will not alter
the sensory properties. While freshness is the main consumer concern on product
quality, subsequent purchases depend upon the satisfaction with texture (Kays, 1999;
Beaulieu and Baldwin, 2002; Kader, 2002; Ngamchuachit et  al., 2014). Toivonen
and Brummell (2008) reported that flesh firmness is a major textural property of
mouth-feel and calcium treatments have been shown to be effective at maintaining
firmness during storage in many fresh-cut fruits. Endogenous and added calcium
can make plant tissue firmer by binding to the pectin carboxyl groups that are gener-
ated through the action of pectin methylesterase (PME) (Conway and Sams, 1983;
Stanley et al., 1995).
Calcium ions can passively diffuse within the cell-wall structure because plant cell
wall porosity is approximately 3.5–9.2 nm (Read and Bacic, 1996), while calcium
28 Advances in Postharvest Fruit and Vegetable Technology

ions are about 0.1 nm (Gillard, 1969). When fruit parenchyma cells are dipped in a
calcium salt solution, calcium ions are primarily transported through the apoplast, or
intercellular spaces, where they are attracted by negatively charged carboxyl groups
in the homogalacturonan that constitutes pectin in the middle lamella and cell wall.
The negatively charged chloride or lactate ions remain unbound in solution (Harker
and Ferguson, 1988; Hasegawa, 2006). There are several ways that calcium could
be applied to fresh-cut fruits such as in conjunction with packaging in low oxygen,
osmotic dehydration, adding to hot water or with irradiation. The combination of a
calcium chloride treatment and packaging with a low O2 concentration was shown
to be more effective than the use of calcium chloride alone to maintain firmness of
fresh-cut “Piel de Sapo” melons (Oms-Oliu et  al., 2010) and “Golden Delicious”
apples (Soliva-Fortuny and Martin-Belloso, 2003). Quiles et  al. (2004) observed
that calcium chloride maintained the structure of “Granny Smith” apples during the
process of osmotic dehydration. (Rico et al., 2007) further showed that warm treat-
ment temperatures (40–60°C) increase the beneficial effects of calcium treatment
due to higher washing solution retention inside the product. Melon pieces dipped
at 60°C in calcium chloride, lactate, or ascorbate showed good firmness retention
(Silveira et al., 2011) as did kiwi fruit dipped in calcium chloride at 45°C (Beirao-
da-Costa et al., 2008). The primary benefit of irradiation was found to be reducing
microbial growth and delaying ripening of fresh-cut fruits and vegetables although
irradiation can cause undesirable textural changes. Prakash et al. (2002) showed that
irradiation substantially decreased the firmness of fresh-cut tomatoes. Magee et al.
(2003) reported that dipping tomato discs in either calcium chloride or calcium lac-
tate solution and exposed to irradiation at 1.25 kGy increased the levels of calcium
and firmness. While Fan et al. (2005) found apple slices treated with calcium ascor-
bate followed by irradiation at 0.5 and 1.0 kGy decreased softening and increased
firmness during storage. In fresh-cut pear cubes, Alandes et al. (2009) showed that
dipping in calcium lactate strengthened the structure of the fruit at microscopic level
by maintaining the fibrillar packing in the cell walls and the cell-to-cell contacts.
Ngamchuachit et al. (2014) also showed that mango cubes that had been dipped in
calcium chloride and lactate were firmer during storage.

2.7 CONCLUSIONS
The application of calcium is a simple and safe technology which has been known
for many years to improve the postharvest performance of a range of fruit and veg-
etables. However, due to increasing consumer trends to minimize the use of synthetic
compounds in foods, calcium treatments are receiving more attention. An important
area of potential application is in reducing microbial growth. However, further stud-
ies are needed to investigate the effects of calcium in reducing bacteria or other
fungi. When calcium is used at an appropriate dosage and timing, it has been shown
to improve product quality and enhance the nutritional value of a range of intact and
fresh-cut produce. Further studies are needed to optimize the uptake of calcium by
managing the form of added calcium and application method. In particular, more
research is required on the effects of calcium in vegetables.
Recent Research on Calcium and Postharvest Behavior 29

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34 Advances in Postharvest Fruit and Vegetable Technology

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Recent Research on Calcium and Postharvest Behavior 35

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 3 Nondestructive
Assessment of
Fruit Quality
Kerry Walsh

CONTENTS
3.1 Introduction.....................................................................................................40
3.2 Available Technologies.................................................................................... 41
3.2.1 Flotation............................................................................................... 41
3.2.2 Load Cell............................................................................................. 41
3.2.3 RGB Camera........................................................................................ 42
3.2.4 X-Ray Radiography and Tomography................................................. 42
3.2.5 Visible and Near Infrared Spectroscopy.............................................44
3.2.6 Time or Spatial Resolved Spectroscopy.............................................. 47
3.2.7 Raman Spectroscopy........................................................................... 47
3.2.8 Fluorescence........................................................................................ 47
3.2.9 Thermal Imaging................................................................................. 48
3.2.10 Magnetic Resonance and Resonance Imaging.................................... 48
3.2.11 Electronic and Chemical Noses........................................................... 49
3.2.12 Force–Deformation.............................................................................. 49
3.2.13 Acoustic Measures............................................................................... 50
3.3 Technologies: In Pack-House........................................................................... 50
3.3.1 Evolution of Technology...................................................................... 50
3.3.2 Weight and Density ............................................................................. 52
3.3.3 Machine Vision Using RGB Cameras................................................. 53
3.3.4 Near-Infrared Spectroscopy................................................................ 53
3.3.5 X-Ray Imaging.................................................................................... 55
3.3.6 Magnetic Resonance............................................................................ 55
3.3.7 Force–Deformation.............................................................................. 56
3.3.8 Acoustic Measures............................................................................... 56
3.4 Technologies: In Field...................................................................................... 56
3.4.1 Automated Harvest and Crop-Load Assessment................................. 56
3.4.2 Estimation of Crop Maturity: DM and Flesh Color
by Spectroscopy................................................................................ 57
3.4.3 Estimation of Crop Maturity: Fluorescence, Volatiles........................ 57

39
40 Advances in Postharvest Fruit and Vegetable Technology

3.5 Technologies: In Transport.............................................................................. 58

3.6 Technologies: In Ripening............................................................................... 59
3.7 Technology Adoption.......................................................................................60
Acknowledgment...................................................................................................... 61
References................................................................................................................. 61

3.1 INTRODUCTION
Noninvasive tools have become increasingly available for medical assessment of
human “internal quality” over the last century, starting with the tool that symbolizes
a medical doctor, the stethoscope. Today, a health assessment may involve noninva-
sive assessment using acoustic (e.g., stethoscope or ultrasound imaging), radiography
(x-ray imaging), magnetic resonance imaging, positron emission tomography, etc. In
this chapter, we explore the relevance and adoption of such technologies for grading
of fruit on quality attributes.
Quality is a concept that has different meanings to each actor in the value chain,
from grower to consumer, depending on the attributes that create value. Attributes of
relevance include those associated with

1. Aesthetic appearance (such as size, weight, external color, external

blemishes)
2. Internal defects (such as tissue browning, including rots, granulation,
translucency)
3. Maturity and ripeness, influencing eating quality (specific gravity, flesh
color [FC], dry matter content [DM], starch content, total soluble solids
[TSS], titratable acidity, flesh firmness, and presence of volatiles)
4. Safety issues (such as the presence of foreign bodies and chemical
contamination)

Ideally, a value chain would assess every item for a selection of attributes relevant
to the produce type (Walsh, 2014).
Large retail chains set specifications for a number of above attributes on a
product basis (e.g., Woolworths, 2014), which generally mirror those set by Codex
Alimentarus (Walsh, 2014). These specifications are set around the needs of a value
chain that requires long shelf-life. Other parts of the value chain, or other value
chains, may give weight to other features. For example, harvest can be delayed to
maximize sugar import, at the expense of firmness and shelf-life. Such a product
targets a market that values eating quality (Figure 3.1). The use of fruit or veg-
etables as nutraceutics (the fusion of “nutrition” with “pharmaceutical,” also often
referred to as “nutraceuticals”) represents a different value chain. Flavonoids, carot-
enoids, anthocyanins, and other bioactive phenolics have a range of claimed health
benefits. For example, acai (palm) fruit of Brazil contains a high level of anthocy-
anin and is marketed for associated health benefits. Logically, such produce should
be graded for content of active ingredient before undertaking value adding activity
(e.g., freeze drying).
Nondestructive Assessment of Fruit Quality 41

FIGURE 3.1  Two ends of the quality spectrum: subsistence mandarin production in Nepal
and Japanese gift fruit, with “light” sorted label (circle at top right, indicating TSS assessed
using near-infrared spectroscopy), with fruit selling at approx. and US$0.70/kg and $40/tray,
respectively.

Thus, the list of attributes associated with “fruit quality” is long, requiring a range
of instrumentation for assessment (quality control) of the varied attributes.

3.2  AVAILABLE TECHNOLOGIES

A seminal review of the range of noninvasive technologies appropriate to the sort-
ing of fruit on internal quality attributes was produced by Abbot in 1999, and other
reviews have updated this assessment (e.g., Ruiz-Altisent et al., 2010). The principle
of these technologies is briefly described in this section, while following sections
highlight current uses of these technologies in orchards, in pack-houses, and during
transport and ripening.

3.2.1 Flotation
The specific gravity of fruit generally increases during maturation as carbohydrate
(dry matter) accumulates. However, the change is slight (0.97–1.04, Abbot, 1999),
such that measurement uncertainties and changes in density due to other reasons, for
example, various fruit disorders, generally render this measure an unreliable matu-
rity index. However, density can be a useful index of disorders such as spongy tissue,
and voids in watermelons. Fruit can be separated based on specific gravity by flota-
tion in a fluid with an appropriate specific gravity. A given application will require
choice of the separation medium, control of medium density and washing/drying of
the fruit. However, for routine grading, calculation of density from fruit weight and
estimated volume is preferred (see below).

3.2.2 Load Cell
The humble load cell is now central to electronic fruit-grading equipment, although
mechanical level/counterweight-balance mechanical grading systems are still pro-
duced. The load-cell systems offer increased speed and accuracy of measurement
42 Advances in Postharvest Fruit and Vegetable Technology

(typically to 1–2 g). Various load-cell technologies exist, for example, hydraulic,

pneumatic and strain, with the “button and washer” type of strain gauge offering the
highest accuracy and precision. Applied force changes the electrical resistance of a
wire, with a typical load cell containing four strain gauges in a Wheatstone bridge
configuration. Cell output is normally in the order of a few millivolts, with an ampli-
fier incorporated into the load-cell design.

3.2.3 RGB Camera
The technology of digital imaging has become ubiquitous (e.g., embedded in phones)
and ever cheaper. These systems are based on silicon charge-coupled device (CCD)
arrays. The CCD elements are sensitive to radiation through the visible range (approx-
imately 400–700 nm) and into the Herschel (or short wave near-infrared) region (to
1100 nm), producing a grey-scale image. In typical RGB cameras, adjacent detector
pixels are coated with a red, green or blue filter, respectively, at the sacrifice of some
image resolution. The red filter commonly employed in cameras allows Herschel-
region radiation to reach the detector. Many cameras employ a short-wave pass filter
(that blocks wavelengths above 700 nm) to produce images based on “visible” light
only. A long-wave pass filter can be used to produce images only in the Herschel
region. A quick test of whether a camera possesses such a filter is to use the camera
to image the operating end of an infrared remote controller—the camera will image a
spot if it does not have a 700 nm long pass filter fitted.
Not only has camera technology surged, but so has machine vision, in terms
of the development of algorithms to identify objects in an image (e.g., review by
Payne and Walsh, 2014). Such systems offer the promise of replacing human labor
wherever routine identification tasks are required, for example, in object counting
or surface color characterization. The image analysis task involves “segmentation”
of the image, that is, classification of those pixels that are associated with fruit, and
“blobbing,” that is, association of fruit pixels into objects (fruit in this case). Typical
features include color, texture (variance between adjacent pixels), shape (perimeter
features) and size (blob pixel count).

3.2.4 X-R ay Radiography and Tomography

X-ray transmission through a material is decreased by absorption and elastic or inelas-
tic scattering. Transmission is thus determined by the photon energy, path length and
average atomic number of the material. The medical fields commonly employ “hard”
x-ray imaging (generator energies >100 kV), but also use “soft” energy x-rays (e.g.,
20–50 kV) for imaging of lower-density material, for example, bones of infants. Dual
energy systems, typified by airport security systems, can detect objects of a range of
densities. “Hard” x-ray radiography is used in fresh-cut salad packaging operations
to detect dense foreign objects, for example, stones. The technology has potential for
the characterization of fruit quality, although fruit density is low (similar to water),
and therefore “soft” x-ray imaging solutions are required. However, tissue density
varies surprisingly little within a fruit (e.g., between skin, flesh and seed), except in
relation to air spaces (voids). Thus, this technique is best suited to the detection of
Nondestructive Assessment of Fruit Quality 43

voids within fruit (Figure 3.2). The first report of the detection of a disorder using
x-ray technology was that of potatoes with hollow “hearts” (Finney and Norris,
1978); commercial adoption followed, based on a simple absorption index rather
than imagery. Other application reports have followed, for example, split-pit peaches
(Han et al., 1992), watercore, rot, insect damage in apple (Schatzki et al., 1997), and
mango spongy tissue and seed weevil (Thomas et al., 1995). These reports involve
imaging, but the recognition rate of (apple) defects by a human operator is poor at
commercial speeds, demonstrating the need for recognition by machines (Schatzki
et al., 1997; Shahin et al., 2001; Mathanker et al., 2011).
X-ray imaging involving computed tomography (CT) based on multiple views of
an object from different directions represents the “high end” of this technology (e.g.,
Figure 3.2). Virtual slices and 3D representation of an object are created, although scan
times of minutes are required (i.e., too slow for in-line sorting) (Zwiggelaar et al., 1997;
Barcelon et al., 1999). In the last two decades, the scientific literature has emphasized
use of x-ray CT, for example, in assessment of tomato maturity (Brecht et al., 1991),

FIGURE 3.2  Images of citrus fruit (a) digital radiograph (with fruit moving at 132 mm/s
using a XR3000 operating at 50 kV, Applied Sorting, Bulleen, Australia), (b) 2 mm 2D
CT-xray of a tray of mandarin fruit, imaged using an accelerating voltage of 35 kV and a
current of 2 mA on a Philips CT system; (c) light scatter from point source (640 nm laser) on
fruit surface; (d) 2D MRI slice. (Adapted from https://www.flickr.com/photos/56604318@
N00/4528898158.)
44 Advances in Postharvest Fruit and Vegetable Technology

water content in apple (Tollner et  al., 1992; Shahin et  al., 1999, 2001), and woolly
breakdown (chilling disorder) in peach and nectarine (Sonego et  al., 1995) (Figure
3.2). Shahin et al. (1999) achieved detection (90% correct, with <10% false positives)
of the internal apple defect, water-core. Microfocus systems have enabled visualiza-
tion of intercellular spaces in small samples (e.g., Verboven et al., 2008). In a quest for
practical application, rather than attempting image analysis of the CT tomograph, some
authors report use of a “CT number.” This number is an x-ray absorption coefficient for
the object. For example, Suzuki et al. (1994) calculated CT numbers for papaya subject
to vapor heat treatment which causes nonripening of internal flesh, while Barcelon
et al. (1999) related CT numbers of mango fruit to moisture content.

3.2.5  Visible and Near Infrared Spectroscopy

Nicolai et al. (2007) and Herold et al. (2009) provide useful reviews of this topic.
The visible (400–700 nm) spectrum of fruit is dominated by pigments such as chlo-
rophyll and carotenoids, while the near-infrared (700–2500 nm) spectra are domi-
nated by absorption associated with the vibration and rotation of O–H and C–H
(dipolar) bonds of organic compounds in water (Figure 3.3). The O–H and C–H

0.4

0.2
Absorbance

0.0

–0.2

–0.4

0.005
Second derivative absorbance

0.000

–0.005

–0.010

400 500 600 700 800 900 1000

Wavelength (nm)

FIGURE 3.3  Absorbance (top panel) and second derivative of absorbance (bottom
panel) spectra of mango fruit collected using interactance optics (Nirvana unit, Integrated
Spectronics, Australia).
Nondestructive Assessment of Fruit Quality 45

bonds produce fundamental frequencies in the infrared (>2500 nm), and a series of

overtone and combination bands in the near-infrared and Herschel (700–1100 nm)
regions. These bands are of progressively decreasing intensity and increasing width
at decreasing wavelengths. The net effect is that infrared wavelengths are absorbed
very strongly and have little effective penetration in biological materials, including
fruit, which are 80%–90% water. Given the greater effective depth of penetration of
the Herschel region (700–1100 nm), this wavelength range is of particular interest
for the assessment of intact fruit, although the absorption features are broader and
overlapped. Further, the Si diode and Si CCD detectors that service this region are
lower in cost than the detectors required for other ranges.
NIRS instrumentation can come in many formats—from full transmission sys-
tems operating at two wavelengths to reflectance-based line-scan hyperspectral
imaging (e.g., Sankaran et  al., 2010). Unfortunately, many scientific publications
appear to involve use of a particular instrument simply because that was available.
Certainly, any scientific publication of the application of near-infrared spectroscopy
to assessment of fruit should document characteristics of the system in use, and jus-
tify its match to the application (as discussed in Walsh, 2005).
Instrumentation that exploits the near-infrared spectrum to assess fruit quality
must address the following design parameters for the assessment of fruit dry matter
or TSS (other criteria can be set for other applications):

1. Wavelength range (typically 700–1000 nm, but up to 2500 nm)

2. Wavelength resolution (typically FWHM 10 nm)
3. Detector signal-to-noise ratio (at least 10,000:1)
4. Light source (typically halogen lamp, light emitting diode or Xe lamp)
stability
5. Repeatability (standard deviation of less than 10 mA on repeated measures
of a white tile)
6. Source-sample-detector geometry (reflectance, partial transmittance, or
complete transmittance)
7. Matching the reference sampling volume to the optically sampled volume
8. Chemometrics (pre-pretreatment of spectra using techniques such as
derivatives, analysis using discriminant analysis or multivariate regression
approaches)

Consider optical geometry: Reflectance geometry, where the detector views an

illuminated region of the sample, is appropriate for an assessment of skin attributes,
such as color, but information will be collected only to a depth of some millime-
ters into the fruit. A partial or full transmittance geometry (i.e., a detector-sample-­
detector geometry between 0 and 180°, but in which the detector does not directly
view an illuminated area of the sample) is appropriate for assessment of an internal
attribute such as flesh dry matter content (DM). In this geometry, the detector views
a non-illuminated region of the sample, such that light reaching the detector has
passed through some volume of the fruit.
Consider wavelength range and resolution: Some spectrometer systems (e.g., CCD
arrays) offer wavelength pixel spacing in the order of 1 nm. This apparent wealth of
46 Advances in Postharvest Fruit and Vegetable Technology

information must be interpreted in the context of the system’s optical resolution (how
broad a line wavelength is when viewed by the spectrometer). Also, the data used
with care as absorbance data of adjacent pixels will be highly intercorrelated and, as
pixel number is high, there is great risk of the over-fitting of regression models. Use
of restricted wavelength ranges can improve the robustness of a model (ability to pre-
dict new sets of data), and allows for the development of lower cost “multispectral”
measurement systems.
The risk of over-fitting multivariate regression models cannot be over-empha-
sized. The scientific literature is full of reports in which groups of fruit were ran-
domly divided into a calibration set and a validation set for model development. Such
a model will predict the attribute of interest within that population, but is likely to
fail spectacularly on a new, independent set. Practical application relies on demon-
stration of the robustness of the model in prediction of independent populations—
following the adage of “no prediction without interpretation, and no interpretation
without prediction.”
Near infrared spectroscopy (NIRS) is, in general, suited to use with thin skinned
fruit (such as apples, stonefruit, and mango), and to the measure of constituents that
present at macro levels in the fruit (Golic and Walsh, 2005). For example, TSS and
DM levels in fruit typically range from 10% to 20%, with a typical error of measure-
ment when using NIRS and intact fruit of around 1% (although the accuracy of TSS
estimation is compromised for samples in which starch levels vary, e.g., in ripening
fruit that convert starch to sugar).
A number of authors have claimed measurement of fruit acidity and firmness.
However, organic acids are present in most fruit at around 1% w/v, and so their near-
infrared signature (of C–H and O–H bonds) will be confounded with that of sugar,
and any correlation with NIRS is likely to be indirect (e.g., higher Brix, lower acid,
with NIRS measurement of Brix) (Subedi et al., 2012). Of course, fruit such as lemon
have acids at high levels and sugars at low levels, so, in this case, direct measure of
acidity by NIRS is possible. Firmness is a physical characteristic, related to cell-
wall composition and structure. NIRS is unlikely to be able to differentiate cell-wall
composition in intact (high-moisture) fruit, so a reported NIRS correlation between
absorption and firmness is likely to represent an indirect relationship (Subedi and
Walsh, 2008). Cell-wall changes may impact light-scattering rather than absorption
coefficients of a fruit.
For example, the detection of “water core” defect and the internal breakdown
defect within apple fruit was first reported by Francis et al. (1965) (R = –0.81 and
0.91, respectively, between defect level and absorbance difference between 740 and
805 nm), based on work with a complete transmission geometry and a single beam
Biospec spectrometer. However, detection of internal breakdown was masked by
internal browning. Upchurch et  al. (1997) reported discrimination of apples with
internal browning (R2 = 0.71, with bruises causing misclassification) while McGlone
et al. (2005) reported a R2 between 0.7 and 0.9 for detection of brown heart disorder
on fruit moving at 500 mm/s. However, such reports are often based on a very limited
population—practical adoption relies on the robustness of the method, for example,
in correct identification of fruit with the disorder in the presence of other, acceptable,
fruit features.
Nondestructive Assessment of Fruit Quality 47

3.2.6 Time or Spatial Resolved Spectroscopy

Beers Law, which related concentration of solute to absorbance, assumes no scat-
tering of light (or at least constant scatter). In practise, the measure of light passage
through fruit involves the measure of an “apparent absorbance,” comprising both
absorbance and scattering features. The amount of scatter may change as cell and
intercellular air-space sizes change.
Two methods have been proposed to allow estimation of the scattering coefficient
(µ′s)—in time-resolved reflectance spectroscopy (TRS), this is estimated from the
time (picoseconds) for a photon to travel through the fruit and to the detector (more
scattering, greater time), while in spatially-resolved spectroscopy it is estimated
from the distance light is scattered from a point source of illumination (e.g., Figure
3.2; Seifert et al., 2015). Given the estimation of a scattering coefficient for a given
fruit, a true absorption coefficient (µa) can also be calculated. Unfortunately, the ini-
tial hope that µ′s might be related to firmness has not been demonstrated.

3.2.7 Raman Spectroscopy
Raman spectroscopy involves inelastic scattering of monochromatic light, with excita-
tion and relaxation of electrons of target analytes. The sample is illuminated with a
(near infrared, visible or ultraviolet) laser beam, and light from the illuminated spot
is dispersed on a monochromator. Wavelengths close to the laser line are due to elas-
tic Rayleigh scattering, and are filtered out, while the rest (Raman scattered light) is
assessed, with the vibrational information specific to the chemical bonds present. Raman
scattering is very weak compared to the intense Rayleigh scattered laser light, and appli-
cation has come only with the relatively recent development of sensitive detectors.
Reported applications include assessment of carotenoid levels, and detection of
pesticides on fruit surfaces (for a review, see Yang and Ying, 2011). For example,
Bicanic et al. (2010) reported use for assessment of carotenoid levels in mango fruit
homogenates. Indeed, Raman spectroscopy has become a method for measuring
carotenoid levels in humans using a through skin measurement, as a biomarker of
fruit/vegetable intake (Scarmo et al., 2012). However, there are no commercial appli-
cations in postharvest operations at this time.

3.2.8 Fluorescence
Fluorescence involves the excitation of a fluorophore with a specific wavelength,
causing excitation of electrons, and emission of a longer wavelength associated with
a drop in electron energy level. Chlorophyll, anthocyanins, carotenoids and flavonols
in intact fruit will fluoresce in the visible range on exposure to UV/violet light. For
example, fruit lycopene content has been related to autofluoresence under excitation
wavelengths of 275 and 400 nm, while chlorophyll concentration is related to emis-
sion at 735 nm given excitation typically around 450 nm. Fluorescence levels are
low, usually below 1% of total absorbed light.
Chlorophyll fluorescence is widely used to assess chlorophyll content and plant
physiological status (stress level) (see review by Maxwell and Johnson, 2000).
48 Advances in Postharvest Fruit and Vegetable Technology

Chlorophyll fluorescence occurs when there is no call for reductive power of the
light reactions, for example, stomata are closed and the dark reactions of photosyn-
thesis do not occur. The ratio of fluorescence at 735 nm and at 700 nm is linearly
proportional to chlorophyll content. Other parameters are calculated from the induc-
tion kinetics for chlorophyll florescence (i.e., the time course of fluorescence from
illumination), such as minimal (Fo), maximal (Fm), and variable (Fv = Fm – Fo) chlo-
rophyll fluorescence. From these values, the potential quantum yield of photosystem
(PS) II, given by the ratio of Fv/Fm is assessed. Fo represents the fluorescence yield
when PSII is able to pass on almost all the electrons excited by light. Fv will vary
depending on the state of photosystem II. Quantum yield (Fv/Fm) is a measure of the
efficiency of the energy transfer process and chloroplast activity. For most species,
the optimal value of quantum yield is 0.83. Stresses like chilling or high-tempera-
ture injuries can reduce PSII function, thereby lowering photochemical efficiency.
In stored fruit, some physiological disorders will result from interruption to PSII
such that chlorophyll fluorescence is high (DeEll and Toivonen, 2003). Measurement
of these parameters can be made using a pulse amplitude-modulated fluorometer,
which involves dark adaption of the sample, followed by a short-duration pulse of
excitation wavelengths, with subsequent measure of fluorescence.

3.2.9 Thermal Imaging
Thermal imaging is commercially used in audits of cool-store insulation efficiency,
or temperature distribution within a cool-room. The wavelengths and intensity emit-
ted by a “blackbody” alter with the temperature of the object, as explained by Planck
relationship. Less than perfect blackbodies have an emissivity coefficient of ≫1;
however biological material with water content above 80% w/w effectively has a high
and constant emissivity factor. For blackbody objects at 30°C, the peak wavelength
of emission is around 10 μm (i.e., infrared). A thermal image is very much a surface
temperature measurement as wavelengths in the order of micrometers are absorbed
very strongly by water-containing objects (such as fruit).
The most accurate thermal imagers use cooled detectors—making for expensive
and power hungry instruments. Typical detector materials include lead sulfide and
mercury cadmium telluride. However, major advances have occurred in the develop-
ment of uncooled detectors, for example, silicon microbolometer arrays, which can
measure a “noise equivalent temperature difference” of 20 mK. Specifications on
such units include: (1) the spectral band sensed by the detector; (2) the detector life;
(3) noise equivalent temperature difference of the detector; (4) minimum resolvable
temperature difference; (5) the number of pixels in the array; (6) the frame refresh
rate; (7) field of view; and (8) power requirements.

3.2.10 Magnetic Resonance and Resonance Imaging

A useful recent review of this topic is provided by McCarthy and Zhang (2012).
Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imag-
ing (MRI) involve use of a radio frequency pulse to excite spin of the nucleus,
with a radio signal emitted as the system returns to equilibrium over a finite
Nondestructive Assessment of Fruit Quality 49

period of time. Signal intensity is recorded over time, providing information

about the environment of the nucleus. Relaxation is generally a function of T1
(spin-lattice or longitudinal relaxation) and T 2 (spin-spin or transverse relaxation).
Information of the bulk sample can be collected (NMR) or 2D or 3D images
can be produced (MRI), and simultaneous measure of a number of fruit quality
parameters is possible.
Various nuclei (e.g., C or P) can be excited to obtain information on chemical
composition (“metabolic profiling”), but most use in fruit imaging has been in terms
of proton mobility (e.g., Figure 3.2d). For example, when H+ nuclei are excited, infor-
mation related to the water content and mobility of the sample can be obtained. T1
and T2 decrease as bulk water becomes viscous, until a point at which T1 increases
again while T2 continues to decrease. Air–water interfaces increase dephasing (the
rate at which the NMR signal decreases), such that T2 can become very short while
T1 is unchanged. Thus images based on T2 relaxation-time can provide information
on the distribution of air spaces within a fruit. 1H NMR sequences have been also
used to study distribution of mobile water with respect to the ripening and defects of
fruit. For some intriguing “fly through” images, see http://i.imgur.com/jTHnQ8W.gif
and http://insideinsides.blogspot.com.au/p/3d-interactive-fruits-and-veggies.html.
However, cost and speed of assessment have limited the adoption of magnetic
resonance imaging. Recent advances in the technology, including improvement in
magnet strength and development of benchtop machines, provide a foundation for
future adoption by the horticultural industry.

3.2.11 Electronic and Chemical Noses

A characteristic aroma is produced by many fruit during ripening, a character that
can be used as an index of ripeness. However, measurement of component volatiles
using chromatographic separation and mass spectroscopy (GC-MS) is technically
demanding and so not suited to application in the horticultural industry. Electronic
noses use an array of electrochemical sensors to create a fingerprint for a given head-
space of atmosphere (e.g., Cyranose 320, Sensigent, California, USA; www.sensigent.
com/products/cyranose.html). However, the sensors used by such systems are prone
to drift, and such systems have not found practical application in the fruit industry.
A simpler technology based on a colorimetric reaction for detecting the presence of
specific volatiles is discussed under the section on “Ripening Technologies.”

3.2.12 Force–Deformation
The firmness of fruit is traditionally destructively assessed with a penetrometer,
involving measurement of the force to push a probe of known diameter at a known
velocity a set distance into the fruit flesh. A range of noninvasive techniques have
been proposed to assess aspects of the firmness, although, as the principles of mea-
surement are different to the penetrometer, different attributes are being assessed
(see reviews by García-Ramos et al., 2005; Khalifa et al., 2011). The search for a
robust noninvasive technique remains something of a holy grail for the horticul-
tural industry.
50 Advances in Postharvest Fruit and Vegetable Technology

One noninvasive estimate of the fruit’s firmness involves measurement of the

micro-deformation of the fruit’s surface under an applied force, or the force required
to deform the surface a set distance. As a variant of this approach, Chen and Ruiz-
Altisent (1996) described an arrangement in which the impact head carried a piezo-
electric accelerometer (which outputs a voltage signal in proportion to applied force),
with the rate of deacceleration of the impact head related to the fruit’s firmness.

3.2.13  Acoustic Measures

A fruit will vibrate when subject to a small force. The response is related to the
modulus of elasticity, mass and shape of the object. The elastic modulus (E; Pa) of
a fruit can be calculated from the highest amplitude resonant frequency (f; Hz) as:

E = C m 2 / 3 f 2 ρ1/ 3

Here, C is a constant related to shape; m, mass of fruit, kg; and ρ, density of fruit,
kg m−3 (Studman, 2001).
The vibration of the fruit will impact on the surrounding air to create an acoustic
signal. This signal can be detected, Fourier transformed, and the dominant (reso-
nant) frequency identified. The movement of the fruit surface can also be assessed
with a single-point laser. Alternately, the resonant frequency(ies) of fruit can be
obtained by vibrating the fruit with a sine wave sweep of a range of frequencies
(e.g., 100–3000 Hz) (e.g., the Vibsoft 4.8, a laser doppler vibrometer from Polytec
Ltd., UK). Another approach was proposed by Sugiyama et al. (1994), in which two
directional microphones were placed at fixed distances away from a point at which a
small impact occurred, and the speed of transmission through the fruit of the domi-
nant pressure wave was assessed. Other technologies have also been proposed, such
as the use of an “air puff” to cause vibration.
Ultrasound systems operate in an inaudible frequency range (around 20 Hz) and
offer potential for the detection and imaging of disorders which result in a change in
fruit density (e.g., Jha et al., 2010). However, the method relies on the presence of a
liquid medium between the transducer and the sample surface, and the signal suffers
high attenuation due to the numerous air–water interfaces in fruit. A useful review is
presented by Mizrach (2008).

3.3  TECHNOLOGIES: IN PACK-HOUSE

This section documents the adoption of noninvasive technologies into the commer-
cial horticultural packing line (Figure 3.4).

3.3.1 Evolution of Technology
The story of the grading of fruit (reviewed by Walsh, 2005) begins with the sorting
of fruit by color, size and shape by humans assessing by sight, and feel. The driver for
mechanization of this sorting process in developed countries has been the increasing
Nondestructive Assessment of Fruit Quality 51

FIGURE 3.4  Example in-line sorting equipment: (a) typical multi-lane electronic grading
platform, with assessment of fruit for color, shape, weight and internal quality parameters;
(b) demonstration unit from MAF Roda, incorporating transmission optics and a visible-
short wave near infra red spectrometer (“InSight 2”) and a four LED multispectral system for
defect sorting (“IDD”); (c) images of orange from the Greefa iPIX, which uses UV illumina-
tion to image skin defects.

cost of labor and the demand for consistency in product quality. Indeed, the develop-
ment of pack-house technology over the last 50 years has been nothing short of spec-
tacular. These developments are poorly reported in the scientific literature, presumably
as the advances have been largely translations of technologies developed in other dis-
ciplines and because they have been driven by private, inhouse development teams.
The first step in the automation of fruit-grading involves human sorters working
alongside a conveyor belt that is carrying fruit, to enhance the efficient flow of the
product. By the early twentieth century, simple mechanical designs were in use to
sort fruit, for example, diverging belts that carry fruit to different positions on the
conveyor depending on fruit size, or screens with slots, graded in size from small to
large, to separate fruit on the basis of diameter. Such systems work well for spheri-
cal fruit. Later came the counterweighted mechanical tipping bucket graders used to
categorize fruit weight.
A large advance occurred in the early 1970s, with the adoption of electronic load
cells to gauge fruit weight more accurately (i.e., to 1–2 g) and more quickly than the
52 Advances in Postharvest Fruit and Vegetable Technology

counterweighted bucket. The load-cell systems involved a shift from a mechanical

to an electronic platform, in which conveyor drop points were electrically actuated.
Other sensor systems can easily be added to an electronic grading platform. By
the late 1970s, RGB camera systems were used to grade fruit on the basis of color.
Conditions within the camera box present a structured environment optimized for
machine vision. For example, uniform lighting can be provided, mirrors can be
used to image the sides of fruit, rollers can be used to rotate fruit passing through
the field of view of the camera, and the background color of the conveyor can be
chosen to maximize contrast with fruit. Such systems can also be used to assess
fruit size. In some systems a second camera operating at Herschel wavelengths (to
1100 nm) was employed to improve the detection of fruit edges and some types of
blemishes.
By the 1990s, more sophisticated algorithms were in use to process images and
categorize features, allowing for detection of blemishes and recognition of a non-
acceptable skin blemish from an acceptable skin mark of stem scar. During the first
decade of the twenty-first century, a range of other sensor technologies were adapted
for application on pack-lines, as discussed below.
As with other industries, with increasing sophistication of the product comes
rationalization of production, with fewer but larger manufacturers of grading equip-
ment with each passing decade. Initially, effectively every production area sup-
ported a local manufacturer with graders of simple design. However, as production
volumes increased, and as market specifications tightened, packing house prefer-
ence shifted to graders of more sophisticated design, that offered more ancillary
equipment (e.g., bin unloading, hydrocoolers, washing or hot dipping, singulators,
drying, bagging, and bar-code labeling for inventory control). Today a multilane
grader is in use, a sophisticated instrument involving investment of hundreds of
thousands of dollars. The internationally dominant manufacturers include MAF,
Greefa, and Aweta P/L.
More recently, grading technologies have begun to move into the field and into use
during fruit transport or ripening. These advances are elaborated in later sections.

3.3.2 Weight and Density

Grading by weight, using load cells (e.g., bending beam load cell), is now a standard
feature of a modern grader. Innovation is required in the application of this technol-
ogy as the fruit cup is moving during measurement, with noise superimposed on
the weight signal from the natural resonant frequency of the load-cell mechanics.
Various solutions have been found to this, generally involving rapid measurement
(e.g., every millisecond), with filtering of higher frequency components.
Density-grading on the basis of specific gravity can be achieved using a solu-
tion of known specific gravity. However, such systems are infrequently used, due to
issues associated with changing solution specific gravity and the need to wash and
dry the fruit. For more spherical fruit, for example, oranges, machine-vision-based
estimates of fruit volume and load-cell measurement of fruit weight can be used to
estimate the fruit’s specific gravity. The extension of such technologies to nonspheri-
cal fruit may become possible as image analysis routines improve.
Nondestructive Assessment of Fruit Quality 53

3.3.3 Machine Vision Using RGB Cameras

As noted above, by the late 1970s, CCD camera systems had been added to fruit
pack-lines, allowing grading of the fruit by size and color. By the early 1990s, more
sophisticated algorithms and procedures were in use, for example, to calculate fruit
volume and, thus, density, given fruit weight, and neural network analyses to deter-
mine whether a given image feature was acceptable (e.g., a fruit stalk) or unaccept-
able (e.g., skin blemishes over more than 2% of fruit surface area). Common features
include the use of conveyor rollers to rotate the fruit while it passes through the
camera’s field of vision, coupled with the use of side mirrors, to image all sides of a
fruit, the use of diffuse lighting and a color for the conveyor that contrasts with the
fruit. Such a structured environment simplifies the machine’s vision task.
A long-wave pass filter on a silicon CCD camera can be used to produce images
only in the Herschel region. Such images are useful for the detection of blemishes,
bruises, or diseased areas on fruit.

3.3.4 Near-Infrared Spectroscopy
NIR spectroscopy based on “point” assessment has achieved a high level of adoption
in the fruit packing industry of Japan and a small level of adoption worldwide. With
a conveyor belt moving at 1 m s−1, a spectrometer integration time of 30 ms incurs
a fruit movement of 30 mm, so a “point” measurement is effectively an average of
at least part of a fruit. These systems typically involve assessment of >200 wave-
length points between 400 and 1000 nm. Typical applications include assessment of
fruit DM and TSS, and certain internal defects. Power is not a limiting factor to the
pack-line application, compared with hand-held assessment, such that temperature
stabilization features for the detector and lamp are often present. Furthermore, as
pack-in-line installations do not experience the range of light level variations encoun-
tered by portable field instrumentation, less frequent referencing is required during
application, which increases the throughput to several pieces of fruit per second.
NIRS technology was first applied to commercial fruit-grading in Japan, from
as early as 1990. The Japanese value chain reward of high-quality fruit (Figure 3.1)
provided the driver for adoption by pack-houses, supported by a range of manufac-
turers such as Fantec, Mitsui, Eminet, and Saika, all of whom based products on a
halogen lamp/photodiode array detector system (Table 3.1). Sumitomo P/L made an
interesting entry to this field in the late 1990s, marketing a system based on the use
of Herschel region diode lasers. The product was subsequently withdrawn from the
market without explanation, and it is a point of conjecture whether this decision was
linked to technical issues. For example, output stability is difficult to achieve with
diode lasers. Further, the much cheaper halogen lamp is a broad emitter, remarkably
effective at producing Herschel region wavelengths, and thus a good alternative to a
laser. For example, a 200 W lamp emitting equally across the range 400–2400 nm
will produce 100 mW over a 1 nm range.
Adaption to the faster grading rates required in Western world agriculture followed
from 2000, with release of equipment by Colour Vision Systems P/L (Australia; now
MAF-Oceania, www.maf.com) and Taste Technology (New Zealand) (Table 3.1).
54 Advances in Postharvest Fruit and Vegetable Technology

TABLE 3.1
Manufacturers of SWNIRS-Based Fruit Grading Units, Claiming Capability
for Assessment of TSS, DM, Flesh Color, Internal Defects Such as Apple
Flesh Browning, Maturity, or Acidity
Company (Location) Website Product/Comment

In-line Application
Aweta (Holland) http://www.inscan-iqa.com/ “IQA”
Eminet (Japan) http://www.eminet.co.jp/web/
hard/index.html
Greefa (Holland) www.greefa.nl “iFA,” “iPIX,” “iQS”
Brettech (Australia) http://www.bret-tech.com.au/ “Hypervision”
MAF (France) www.maf.com “InSight,” “IDD”
Mitsui (Japan) http://www.mitsui-kinzoku.
co.jp/en/seihin/s_sozai/other/
Saika (Japan) http://www.saika.or.jp/aguri/
seihin.html (in Japanese)
Sacmi (Italy) www.sacmi.it Sumitomo Ceased production diode laser-based
(Japan) system for melon TSS
Taste Tech—Compac www.taste-technologies.com T1 R2 M2
(NZ)

Hand-Held (In-field) Application

Astem (Japan) http://www.astem-jp.com/
english/products.html
Fantec (Japan) No longer trading “NIR Gun”; early innovator in this field
Felix instruments www.felixinstruments.com “F750,” operating principles of Nirvana
(USA)
Force-A (France) www.force-a.eu “Multiplex” absorbance and fluorescence
spectroscopy, also in tractor-mounted
format
Integrated spectronics No longer trading “Nirvana”
(Australia)
Sunforest (Korea) http://sunforest.en.ec21.com/ “H-100C,” newest market entrant

Note: Location is of company headquarters.

Other grader manufacturers followed, for example, Greefa (Netherlands), Aweta

(Netherlands) and Unitec (Italy). Interestingly, while the Japanese manufacturers of
NIRS equipment for fruit-grading were independent of the conveyor manufacturers,
in the Western world it is the grader-manufacturers themselves that have developed
NIRS products. The list of manufacturers in Table 3.1 is not complete, with new
entrants to this field occurring continually. However, rationalization of the field is
also expected, as seen in all manufacturing fields in a global economy.
Equipment design differs in terms of light sources (halogen, LED, laser), opti-
cal geometry (complete or partial transmission, or reflectance), detector figures of
Nondestructive Assessment of Fruit Quality 55

merit (such as wavelength resolution, wavelength range, pixel resolution, signal to

noise, stray light), and chemometrics (such as multiple linear regression, partial least
squares, discrimination analysis).
Recently, focus has shifted from the capability to sort by “positive” quality attri-
butes such as DM and TSS, to sorting by “negative” attributes (internal defects). For
example, a number of commercial providers of sorting equipment (Table 3.1) claim
detection of water content and browning in apple, internal browning in kiwifruit,
and granulation in citrus and clementine. All such systems are based on transmission
spectroscopy, but there is no characterization of the systems or validation of their
efficacy in the scientific literature.
Multispectral and hyperspectral imaging are further levels of sophistication that
have yet to find commercial application in fruit grading (Sankaran et  al., 2010),
although a range of hyperspectral systems are commercially available (e.g., www.
zolix.com.cn/en/prodcon_370_375_359.html). Exceptions include the HyperVision
in-line grading system (Table 3.1), which acquires reflectance hyperspectra using a
line-scan camera (charge couple device or CCD) operating over the spectral range
of 450–1150 nm and 6 × 300 W tungsten halogen lamps, with a distance of 800 mm
from the CCD to the conveyor belt (e.g., Wedding et al., 2011), and the Helios (Helios,
http://www.hyperspectral-imaging.com/products.html) “chemical imaging” system
used in detection of “sugar end” in potato.
A converse trend is to produce equipment capable of assessing only a few wave-
lengths, reducing the cost and complexity of instrumentation. This type of example
often relies on the use of LEDs, now available at a range of wavelengths through
the 400–1100 nm spectrum. For example, the “DA-head” (Turoni, Italy) provides a
measurement of fruit chlorophyll content, based on absorption at two wavelengths
around the chlorophyll peak, with the claim that segregation of harvested fruit (e.g.,
peach, nectarine) into homogeneous classes of ripening can be achieved, at a speed
of 4–5 fruits/s. In another example, the “IDD2” unit (MAF, France) utilizes four
wavelengths in assessment of internal defects such as diffused browning of the apple.

3.3.5 X-Ray Imaging
Line-scan systems allow the acquisition of transmission images of fruit passing on
a conveyor. The best and perhaps only commercial application of this technology
in the context of intact produce is the assessment of potato tubers for the presence
of internal voids, based on the seminal work of Finney and Norris (1978). Several
manufacturers service this market (e.g., BEST, 2014). The potential health hazards
and cost of x-ray imaging are limits to the adoption of this technology.

3.3.6 Magnetic Resonance
Cost and speed of assessment have limited the adoption of magnetic resonance imaging.
However, recent advances in magnet technology have allowed increased field strength
at reduced cost (Zhang and McCarthy, 2013). For example, Aspect Imaging (Israel)
offers a prototype magnetic resonance imaging scanner coupled with a conveyor belt
for measuring fruit quality attributes in-line, sorting fruit in groups of 10–12 fruits/s.
56 Advances in Postharvest Fruit and Vegetable Technology

3.3.7 Force –Deformation
The Sinclair (2014) “iQ” technology is an impact system with an accelerometer,
assessing the deceleration rate of a light object striking the surface of the fruit. An
air-actuated bellows (as used in the Sinclair fruit labeling system) carries the accel-
erometer into contact with the fruit. Four measurements of the one piece of fruit are
made as it travels down the conveyor.
The Greefa “iFD” (“intelligent Firmness Detector”) on-line firmness-testing unit
operates on a similar principle, with a set of sensor heads that impact the top of the
rotating fruit, taking up to 20 measurements around each fruit. Effective use with
apples, avocados, mangos, peaches, and kiwis has been claimed (Greefa, 2014).

3.3.8  Acoustic Measures

Given the level of vibrations present in an operating grading machine it is perhaps
not surprising that no acoustic-based technology has achieved commercial success
in on-line grading. For example, Aweta (www.aweta.com) offers equipment to mea-
sure the resonant frequency of vibrations within a fruit produced by a light tap of the
fruit. This equipment is available in a benchtop format and is not available in on-line
applications, in which environmental vibrations must complicate use.

3.4  TECHNOLOGIES: IN FIELD

3.4.1  Automated Harvest and Crop-Load Assessment
The last half century has seen great adoption of technology within the pack-house,
driving labor costs down. The logical next areas for attention are in reduction of
harvest costs (which account for roughly 40% of the orchard’s operating costs) and
in moving aspects of the sorting function of the pack-house into the field. The first
steps in this direction have appeared as harvest aids manned by crews that per-
form sorting functions in the field. Automated harvesters are not yet commercially
viable for fleshy fruits, but their development can be expected to continue, spurred
by development of ancillary devices, for example, the vacuum-assisted harvest
device (US patent 4,501,113; Brown, 2014). Another target for automation is the
assessment of fruit load per tree. Ideally, such a system would allow identifica-
tion and counting of fruit on a per tree basis, estimation of fruit size, recording of
fruit location on a tree to allow estimation of increase in fruit size between repeat
measurement events, and data warehousing of information to allow comparison of
yields per tree across years.
Automation of these orchard tasks is dependent on machine vision systems that
allow recognition of fruit within the “unstructured” and variably illuminated envi-
ronment of the tree canopy (e.g., see review by Payne and Walsh, 2014). The default
sensor for a machine-vision system is a RGB camera, given comparative cost and
availability, but other technologies also have merits (e.g., LIDAR, time of flight
imaging). Any action to improve the “structure” of the environment will improve the
machine-vision result, for example, use of production systems that present fruit in 2D
Nondestructive Assessment of Fruit Quality 57

arrays, or night imaging with use of artificial illumination, in which the background
is effectively removed.
Of course, some applications will be more difficult than others. The detection
of green fruit set amidst a green canopy will be difficult, compared to colored fruit
arranged on a plane surface. For example, in kiwifruit orchards, the kiwifruit hang
suspended under vine canopies, and the fruit contrast in color with the foliage.
Wijethunga et al. (2008) reported automated counting with accuracy levels >90% for
a gold kiwifruit image data, with CIE Lab color space input.

3.4.2 Estimation of Crop Maturity: DM and

Flesh Color by Spectroscopy
Indices of fruit maturity should be easy to assess, objective and preferably nondestruc-
tive, informing decisions on timing of harvest. In some fruit, external color and shape
are adequate measures, but in others this is not the case. Other indices include tem-
perature integrals (degree days), flesh color, skin and flesh pigmentation (e.g., decrease
in skin chlorophyll and increase in skin carotenoid and anthocyanin concentration).
Several technologies are available for in-field use. Various absorption indices
involving assessment of fruit on tree at several wavelengths have been proposed. For
example, the Pigment Analyzer (PA1101, CP, Germany) has been used to assess shift
in the chlorophyll red edge position (the wavelength at which the first derivative of
the chlorophyll peak is maximal) as fruit mature (Zude, 2003; Seifert et al., 2014,
2015). The “DA meter” (Turoni, Forli, Italy) calculates the difference of absorption
at 720 and 680 nm (i.e., indexes chlorophyll content). The DA index has been related
to stonefruit maturation (Ziosi et al., 2008), although the index can be varied and
site-specific (e.g., sun exposure can cause loss of chlorophyll).
Two hand-held Herschel region spectroscopy instruments have been applied to the
assessment of mango fruit quality attributes: the FT20 (Fantec, Japan) (Saranwong
et al., 2003) and the Nirvana (Integrated Spectronics, Sydney) (Subedi et al., 2007)
(Table 3.1, Figure 3.5). For example, Subedi et al. (2007) reported on the use of a
hand-held spectrometry system to assess mango fruit DM and internal flesh color, in
the context of informing harvest scheduling, by

1. Assessing variation within a given tree canopy.

2. Defining areas within an orchard containing fruit of similar maturity.
3. Repeated measures over time to provide information on the rate of fruit
maturation (e.g., Loefflen and Jordan, 2013, report use of such data in pre-
dictive modeling in kiwifruit and mango supply chains).

The value of such technology is achieved when used to reduce variability in fruit
maturity or ripeness.

3.4.3 Estimation of Crop Maturity: Fluorescence, Volatiles

FORCE-A (Orsay, France) markets a fluorescence-based optical sensor
(MULTIPLEX®) suited to in-field fruit measurement of anthocyanin, chlorophyll
58 Advances in Postharvest Fruit and Vegetable Technology

FIGURE 3.5  Example hand-held devices: (a) acoustic device measuring time delay between
impact to fruit and detection of resonant vibration by microphone at a known distance
from impact; (b, c) hand-held near-infrared spectrometers for fruit TSS or DM from Felix
Instruments (USA) and Fantec (Japan), respectively; (d) benchtop device for assessment of
acoustic resonant frequency from Aweta (Holland), and (e) fluorometer for anthocyanin and
chlorophyll assessment, from Force-A (France).

and flavonol contents as maturity and quality indices (Figure 3.5). For example, use
of this technology to monitor grape and oil palm fruit maturation by anthocyanin
accumulation was reported by Ghozlen et al. (2010) and Hazir et al. (2012), respec-
tively, while assessment of kiwifruit quality and maturity of apples, based on flavonol
content, was reported by Pinelli et al. (2012) and Betemps et al. (2011), respectively.
This technology is also available as a mounted sensor for agricultural machines.
Cyranose (USA) offers an electronic nose for volatile assessment as an alternate
method for assessment of fruit maturity (Figure 3.6).

3.5  TECHNOLOGIES: IN TRANSPORT

Wireless networks allow transfer of information—allowing data access to instru-
mentation placed in orchards and in transport systems. A useful review of the current
state of this developing technology is provided by Ruiz-Garcia et al. (2009).
Nondestructive Assessment of Fruit Quality 59

FIGURE 3.6  (a) Pears in clamshell with chemical sensing labels (red spot changes from red
to yellow as fruit ripens (RipeSense, www.ripesense.com); (b) Cyranose320 electronic nose
(Sensigent, USA http://www.sensigent.com/products/cyranose.html) in trials for assessment
of grape maturity (http://www.vt.edu/spotlight/innovation/2013-12-09-nose/M_device.jpg).

Fruit temperature control during transport and storage is critical to the posthar-
vest shelf-life. Unfortunately, information on breakdowns in the cool chain has in
the past only been available “after the fact.” The use of temperature-logging devices
within loads and wireless networking offers potential for a new level of management,
including manipulation of ripening during transport, on the basis that quality can be
predicted from temperature history. For example, Stepac P/L offers “Xsense”—a
wireless network of temperature and relative humidity sensors placed within pal-
lets of a consignment coupled to a GPS device, with data relayed to a centralized,
web-based database that can model remaining shelf-life (http://www.po.stepac.
com/; https://www.xsensesystem.com/doa 14/2/14). Managers at different locations
can access the system, which can be set to issue alerts, for example, once threshold
temperatures are exceeded.

3.6  TECHNOLOGIES: IN RIPENING

Controlled ripening of climacteric fruit requires control of temperature and a sys-
tem to introduce ethylene. Fruit are typically monitored for their stage of ripening
as assessed by skin or flesh color, or firmness, with the aim of moving the fruit
while a reasonable shelf-life still remains. Several measurement technologies are
available for installation into sealed storage or ripening chambers, allowing for con-
tinuous measurements, while other technologies are available in an “at-line” form,
for measurement of representative sample numbers of fruit. Continuous monitor-
ing systems rely on assessment of representative fruit within the consignment. For
example, HarvestWatch P/L offers a system for continuous monitoring of chloro-
phyll fluorescence of stored product for dynamic controlled atmosphere storage of
fruits and vegetables (http://www.harvestwatch.net/doa. 14/2/2014). Installation in
>1000 controlled atmosphere rooms in >15 countries, storing >200,000 t of apple,
is claimed. The DAFL (Difference Absorbance Fruit Logger, Turoni P/L, Italy) is
60 Advances in Postharvest Fruit and Vegetable Technology

another device for continuous monitoring, in this case, based on the difference of
absorption at 720 and 680 nm, with daily transmission of results to a central server
outside the cold-storage room.
Electronic noses also have potential for assessment of ripening, but commercial
adoption has not occurred. In contrast to the chlorophyll and volatile assessment
technologies, RipeSense P/L (New Zealand; www.ripesense.com) offers a “simple”
volatile detection technology based on a colorimetric reaction for specific volatiles.
This technology is incorporated into a label and included within packaging material
(Figure 3.6). A “clamshell” packaging is used that allows gaseous compounds to
accumulate, as well as providing protection to the fruit. The first commercial applica-
tion of this system was for pears.
Several “at-line” noninvasive measurement technologies for firmness have seen
limited commercial use at-line in ripening rooms. Each of these techniques mea-
sure parameters linked to mechanical properties of fruit, although each measures
somewhat different properties, and all are subtly different from that measured by a
penetrometer.
Agro Technologie (Les Eaux, France) offers a micro-deformation device, the
“Durofel,” which uses a flat-ended probe (three sizes, matched to fruit type). Its
use with apricots, tomatoes, cherries and other soft fruits has been reported (Agro
Technologie, 2014). Technology marketed by Aweta (Acoustic Firmness Sensor, or
AFS; Aweta, 2104) is based on measurement of the resonant frequency of sound
emitted by a fruit following a light tap. It is recommended for measurement of the
firmness of apples and tomatoes. Technology developed by Sugiyama et al. (1994)
and Subedi and Walsh (2008) evaluates the velocity of a pressure wave travelling
through the fruit from the point of impact of a low force (Figure 3.5). The Sinclair
accelerometer system (as used on-line) has also been available in an “at-line” format.

3.7  TECHNOLOGY ADOPTION

In this chapter we have reviewed the applicability and availability of a range of
instrumentation for nondestructive assessment of fruit quality. Adoption of this tech-
nology will be driven by need—when a fruit quality issue becomes a severe problem
for marketability, there is a driver for uptake of a relevant technology. Of course, as
with any commercial decision, a sound cost-benefit analysis is required for use of
a given sorting technology. For example, consider a packing house operating for a
harvest period of 100 days per annum, packing 10 t per lane per day or 1000 t of fruit
over the season and selling fruit at $1/kg. If the cost of a piece of sorting equipment
is US$30,000 pa (asset value US$80,000 leased or depreciated at US$20,000 per
annum, plus operator and maintenance costs), then a cost of US$0.03/kg is incurred.
Thus the cost of such technology is not trivial but neither is it prohibitive. The extra
cost/complexity of such a system can only be justified where a clear supply chain
advantage exists. For example, based on the figures above, a farm-gate value of pro-
duce packed in a single day by a single pack line is US$10,000. The rejection by a
supply chain of eight days of production because of presence of a defect incurs a
financial loss equivalent to the cost of the technology.
Nondestructive Assessment of Fruit Quality 61

Noninvasive sorting capabilities can be used to reinforce an existing marketing

system, for example, to meet specifications, or to realize “disruptive” applications,
opening new markets. The introduction of new technologies also creates different
opportunities—for example the range of technologies employed in the value chain
may reduce the need for physical labor but increase the requirement for a range of
professionally trained staff (e.g., in information technology and mechatronics).
Let us finish this chapter with a case study: the adoption of an NIRS technology
into a supply chain. Harvest Fresh P/L attempted implementation of in-line Brix and
DM sorting systems in the early 2000s, marketing lines of fruit that more than met
retailer specifications on these attributes. However, premiums were not consistently
offered by retailers, such that the extra costs incurred in growing to and sorting for
higher specifications was not economic. Instead, adoption of the technology occurred
in support of quality control for a Plant Variety Rights protected variety. The fruit
of this variety presented well in terms of skin color but the normal visual clues on
fruit maturity were not present—leading to regular harvesting of immature fruit
with less than ideal eating quality. A quality control system was implemented using
hand-held near-infrared spectrometers (Nirvana units from Integrated Spectronics,
Sydney) with monitoring of fruit for dry matter content in all orchard blocks on a
weekly basis in the lead up to harvest. The quantile-functions approach developed by
Loeffen and Jordan (2013) was implemented in a decision support system, managed
across all plantations, in which the percentage of fruit under specification for a DM
criterion is monitored and modeled, allowing estimate of the optimal harvest date.
The system allows an estimate of appropriate harvest date, supporting decisions on
contracts for harvest labor, fruit transport, and marketing. Further, as DM at matu-
rity is linked to Brix at fully ripe in this climacteric fruit, fruit of high eating quality
are also produced.

ACKNOWLEDGMENT
Support of Horticulture Australia Ltd., Hortical P/L, MAF Oceania P/L, Integrated
Spectronics P/L, and OneHarvest is acknowledged, and Bed Khatiwada for input in
the preparation of tables.

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 4 Biological Control of
Postharvest Diseases
Giuseppe Lima, Simona Marianna Sanzani,
Filippo De Curtis, and Antonio Ippolito

CONTENTS
4.1 Introduction..................................................................................................... 65
4.2 Antagonist Microorganisms as Biocontrol Agents..........................................66
4.3 Role of Microbial Ecology in Postharvest Diseases........................................ 68
4.4 Influence of Agrochemicals on Useful Microbial Populations of
Phyllosphere and Carposphere........................................................................ 69
4.5 Integration of Antagonist Microorganisms with Other Control Systems.......... 70
4.5.1 BCAs and Physical Means................................................................... 70
4.5.2 BCAs and Natural Compounds........................................................... 72
4.5.3 BCAs and Low Dosage of Synthetic Fungicides................................. 74
4.5.4 Preharvest Application of BCAs......................................................... 75
4.5.5 Multifaceted Approaches..................................................................... 77
4.6 BCA Formulation............................................................................................ 77
4.7 Genomic Approaches to Study BCA–Host–Pathogen Interactions................. 78
4.8 Concluding Remarks....................................................................................... 80
References................................................................................................................. 81

4.1 INTRODUCTION
Postharvest diseases are responsible for consistent losses of fresh fruits and veg-
etables, with up to 50%–60% of fresh produce discarded because of postharvest
spoilage. Pathogenic microorganisms, such as fungi, mainly belonging to the genera
Alternaria, Aspergillus, Botrytis, Fusarium, Monilinia, Penicillium, Rhizopus, are
the main causal agents of postharvest deterioration (Snowdon 1990). Some of these
pathogenic fungi such as Alternaria alternata, Aspergillus spp., Fusarium spp. and
Penicillium expansum also produce toxic metabolites known as mycotoxins.
Despite the use of modern storage facilities and techniques, synthetic fungicides
are still the main control means for reducing rots over extended periods of storage
and/or transportation. However, increasing global concerns about environmental and
human health risks due to pesticide residues has consistently reduced their use in the
field near harvest and/or in postharvest situations (Droby et al. 2009). The use of
synthetic fungicides is also discouraged by the increase of fungicide-resistant patho-
gen strains in packing houses as a consequence of their prolonged and intensive use

65
66 Advances in Postharvest Fruit and Vegetable Technology

(FRAC 2013), the very low or even “zero” chemical residues required by fruit retail-
ers and consumers (Cross and Berrie 2008), as well as more restrictive international
regulations (e.g., EC Directive 2009/128).
Such issues, jointly with the latest worldwide guidelines on integrated and sus-
tainable diseases management, is increasing research efforts to find more environ-
mentally acceptable and safer measures for controlling postharvest disease. Use of
natural bioactive compounds, microbial antagonists, and physical means are the
main approaches for finding new solutions to ensure fruit quality and safety (Palou
et  al. 2008; Mari et  al. 2009). Research with microbial biocontrol agents (BCAs)
in pre and/or postharvest situations of fruit and vegetables started around 30 years
ago (Wilson and Pusey 1985) against brown rot (Monilinia fructicola) of peach
with a strain of Bacillus subtilis. This line of research is still of intense worldwide
interest (Janisiewicz and Korsten 2002; Ippolito et  al. 2004; Lima and De Cicco
2006; Wisniewski et al. 2007; Droby et al. 2009; Liu et al. 2013). The high amount
of research carried out in this period (with more than one thousand such scientific
articles published!) has shown the effectiveness of a large number of BCAs against
numerous and harmful postharvest pathogens (Lima et al. 1999; Ippolito and Nigro
2000; Janisiewicz and Korsten 2002; Liu et al. 2013). However, BCAs, when applied
as stand-alone treatment under commercial conditions, sometimes fail to control
postharvest pathogens at a satisfactory level and this is a major obstacle for their
large-scale implementation (Droby et al. 2009; Janisiewicz 2013).
In the present chapter, we supply fundamental information on (1) the basis of
biological control of postharvest diseases; (2) the nature and characteristics of the
most studied BCAs; (3) key strategies for improvement and optimized use of BCAs
formulation; (4) constraints and obstacles to be overcome for the effective and large-
scale implementation of BCAs; and (5) future perspectives in the biological control
of postharvest diseases.

4.2  ANTAGONIST MICROORGANISMS AS BIOCONTROL AGENTS

In the biological control of postharvest diseases, the natural epiphytic microflora of
the surfaces of fruit and vegetable is the main source for selection of BCAs (Droby
et al. 2009; Liu et al. 2013). The main antagonists selected include bacteria, yeasts,
and yeast-like fungi.
The most investigated biocontrol bacteria are Bacillus subtilis (Wilson and Pusey
1985), Pseudomonas cepacia (Janisiewicz and Roitman 1988), P. syringae (Bull
et al. 1997), Erwinia herbicola (Bryk et al. 1998), Pantoea agglomerans (Usall et al.
2008), B. amyloliquefaciens (Arrebola et al. 2010), B. megaterium (Kong et al. 2010),
P. aeruginosa (Shi et al. 2011), Citrobacter freundii (Janisiewicz et al. 2013). The
main mechanisms of action exerted by selected biocontrol bacteria are (1) secretion
of bioactive molecules such as antibiotics and cell-wall degrading enzymes (Ongena
and Jacques 2008); (2) stimulation of the plant’s defensive capacity (Jones and Dangl
2006); and (3) competition for space and nutrients (Poppe et  al. 2003). In several
studies, selected biocontrol bacteria, in addition to competition for space and nutri-
ents, exerted a strong inhibitory effect on fungal pathogens by affecting conidial
Biological Control of Postharvest Diseases 67

germination and/or germ tube elongation through production of bacterial lipopep-

tides such as fengycins, iturins, and surfactins (Ongena and Jacques 2008).
Currently bacterial-based products available for commercial use in pre- and
­postharvest operations are (1) BioSave® (JET Harvest Solutions, Longwood, FL,
USA), which is based on P. syringae as an active ingredient and is used for the control
of sweet potato and potato diseases; (2) Serenade® (Bayer, Leverkusen, Germany),
based on B. subtilis and used against pre- and postharvest diseases of stone and
pome fruits, strawberry, and tomato; (3) Pantovital® (Domca, Granada, Spain), based
on P. agglomerans and used against pre- and postharvest diseases of different fruit;
and (4) Amylo-X® (Biogard CBC, Grassobbio, Italy), based on B. amyloliquefaciens
and used against fungal and bacterial diseases of different vegetables.
Concerning yeasts, numerous species have been isolated from a variety of sources,
including fruit and leaf surfaces, soil, and seawater, and their potential as BCAs
has been extensively investigated (Droby et  al. 2009; Liu et  al. 2013). The most
studied yeasts are Pichia guilliermondii (Wilson et al. 1993), M. pulcherrima (De
Curtis et al. 1996), Candida oleophila (Lima et al. 1997), Saccharomyces cerevisiae
(Mari and Carati 1997), C. laurentii and Rhodotorula glutinis (Lima et al. 1998),
C. saitoana (El Ghaouth et al. 2000), P. membranaefaciens (Fan and Tian 2000),
C. sake (Nunes et al. 2001), Metschnikowia fructicola (Kurtzman and Droby 2001),
Debaryomyces hansenii (Hernández-Montiel et al. 2010), Rhodosporidium paludi-
genum (Wang et  al. 2010), R. kratochvilovae (Castoria et  al. 2011), Cryptococcus
humicola (Bonaterra et  al. 2012), Kloeckera apiculata (Bonaterra et  al. 2012),
Cystofilobasidium infirmominiatum (Vero et al. 2013).
The main mechanisms of action of antagonist yeasts are (1) competition for space
and nutrients; (2) activation of host defences; (3) secretion of bioactive molecules
such as antibiotics and cell-wall degrading enzymes; (4) direct physical interaction
with fungal hyphae; and (5) tolerance of the biocontrol yeast to reactive oxygen
species (ROS) produced in the fruit in response to wounding (Castoria et al. 2003;
Droby et al. 2009).
Inspite of the high number of selected strains, very few yeast-based commer-
cial biofungicides are available for pre- and/or postharvest use. These include (1)
YieldPlus® (Lallemand, Montreal, Canada), based on Cryptococcus albidus; (2)
Candifruit® (IRTA, Lleida, Spain), based on C. sake; 3) Nexy® (BioNext, Paris,
France), based on C. oleophila; and 4) Shemer® (Koppert, The Netherlands) based
on M. fructicola.
Research on yeast-like fungi includes studies on Aureobasidium pullulans (de
Bary) Arnaud, which is effective in preventing postharvest fungal diseases of sev-
eral crops in the field and in postharvest conditions (Leibinger et  al. 1997; Lima
et al. 1997, 1999, 2003). Of great interest is the use of A. pullulans isolates during
flowering, a phase in which some necrotrophic pathogens can attack the host tis-
sues, as evidenced by Lima et al. (1997) using strain L47 of A. pullulans for pre-
venting ­postharvest grey mold (B. cinerea) rots on strawberries. A. pullulans also
proved to be a very effective antagonist when used in orchard trials in combination
with reduced rates of fungicides to control rots and extend the shelf-life of apples
(Leibinger et al. 1997; Lima et al. 2003).
68 Advances in Postharvest Fruit and Vegetable Technology

The main modes of action reported to play an important role in the biocontrol
activity of A. pullulans isolates are (1) competition for space and nutrients; (2) stimu-
lation of host defences (Castoria et  al. 1997); and (3) production of extracellular
depolymerase enzymes such as chitinases and glucanases, which act on pathogen
cell-walls (Castoria et  al. 2001). Currently, there are three commercial products
based on A. pullulans for use from the flowering to the postharvest stages: (1) Boni
Protect®; (2) Blossom protect®; and (3) Botector® (Bioferm, Tulln, Austria).

4.3  ROLE OF MICROBIAL ECOLOGY IN POSTHARVEST DISEASES

While the aerial plant surface is not an ideal environment, it is populated by a vari-
ety of epiphytic nonpathogenic microorganisms playing a central role in different
ecological interactions. The epiphytic microbial communities include different gen-
era of bacteria, yeasts, yeast-like fungi, and filamentous fungi. Bacteria, the most
abundant inhabitants of phylloplane and carpoplane, differ among plant species, as
per the growing season, and the physical and nutritional conditions of plant surface
(Lindow and Brand 2003). Yeasts (white and pink yeasts) and yeast-like fungi are
widespread and active phylloplane and carpoplane colonizers, effective natural buf-
fers against phyllosphere and/or carposphere plant pathogens (Andrews and Harris
2000). Antagonists of postharvest pathogens have also been isolated from other
sources, as roots, soil, and seawater (Liu et  al. 2013). Yeasts and yeast-like fungi
seem ideal candidates for an efficient biocontrol of postharvest pathogens since they
colonize efficiently and steadily wounded and nonwounded plant surfaces, even under
unfavorable conditions (Lima et al. 1998; Ippolito et al. 2005; De Curtis et al. 2012).
The size and composition of epiphytic microflora populations depend on the host
species, nutrients, rainfall, humidity, and temperature, but are also influenced by
human activities such as fertilizer and pesticide application (Nix-Stohr et al. 2008;
Droby et al. 2009). Moreover, the growth of the epiphytic microflora is supported
by a variety of organic compounds, as vegetal exudates and pollen and honeydew
deposits. Epiphytic microorganisms are able to deplete from plant surfaces nutrients
essential for both growth of fungal pathogens and beginning of infection process
into vegetal tissues. Yeasts and yeast-like-fungi, in general, are tolerant to low nutri-
ents concentration and are able to utilize and metabolize various carbon sources,
inorganic and organic nitrogen, lipids and other exogenous nutrients like pollen and
aphid honeydew (Nix-Stohr et al. 2008).
The type and availability of nutrients affect the microbial population present on
the phylloplane (Andrews and Harris 2000; Nix-Stohr et al. 2008). In general, all
yeasts require sources of carbon, nitrogen, minerals, and vitamins for maintenance
and growth, and this need varies considerably among yeast species. Yeasts preferen-
tially utilize simple sugars versus polysaccharides, proteins, and lipids. Nevertheless,
some yeasts isolated from phylloplane are tolerant to very low nutrient concentration
(Nix-Stohr et  al. 2008) and, in particular conditions of starvation, they can show
high and specific extracellular enzymatic activities (e.g., chitinases and glucanases),
which depolymerize the cell-wall structure of fungal pathogen to metabolize the
derived simple carbon compounds (Castoria et al. 1997, 2001). Several yeasts used
as BCAs were isolated from samples of a variety of fruit and vegetables or even
Biological Control of Postharvest Diseases 69

from other environmental matrices as seawater and soil (Liu et al. 2013). As regards
yeast-like fungi, A. pullulans is the predominant and widespread organism in vari-
ous environments (Blakeman and Fokkema 1982).

4.4 INFLUENCE OF AGROCHEMICALS ON USEFUL MICROBIAL

POPULATIONS OF PHYLLOSPHERE AND CARPOSPHERE
The term agrochemical in this context mainly includes biocide products as fungi-
cides, bactericides, acaricides, herbicides, insecticides, and nematocides. Knowledge
of the effects of commonly used agrochemicals on nontarget saprophytic microflora
of plant surfaces, as well as on selected antagonist microorganisms, is essential for
efficient management of postharvest diseases from an integrated point of view. This
information can identify agrochemicals with or without a negative impact on useful
nontarget epiphytic microorganisms as well as on select BCAs.
Since the target pathogens responsible for postharvest diseases are mainly
fungi, particular attention is given to interactions with fungicides. The compat-
ibility of BCAs with fungicides is a key prerequisite for their successful use in
integrated pest management schedules, since the survival and colonization of a
BCA after its introduction into the hard niche of the phylloplane may be influ-
enced by their interaction with chemicals. The effect of fungicides on naturally
occurring saprophytic microorganisms was assessed in various studies (Dik and
van Pelt 1992; Southwell et al. 1999; Buck and Burpee 2002; Lima et al. 2003;
Cadez et al. 2010; De Curtis et al. 2012). For instance, Dik and van Pelt (1992)
observed that the fungicides prochloraz and triadimenol had no effect on pink
and white yeasts, while maneb drastically inhibited the yeasts growth; they also
observed that a combination of insecticides with fungicides caused a reduction
in the presence of honeydew on the phyllosphere and, consequently, drastically
reduced the presence of yeasts.
Recently, Debode et al. (2013) observed that the population dynamic of the main
epiphytic yeast species on strawberry leaves and fruit was not affected by the appli-
cation of the fungicides cyprodinil, fludioxonil, boscalid, and pyraclostrobin. These
results are in agreement with those of Cadez et  al. (2010) who observed a higher
presence of yeasts on the surface of the berries of grapes treated with the com-
monly used fungicides iprodione, pyrimethanil, and cyprodinil plus fludioxonil.
Conversely, Buck and Burpee (2002) showed that the combination cyprodinil plus
fludioxonil on epiphytic yeasts of grapes and grasses resulted in a dramatic reduc-
tion in yeast density. Our recent studies evidenced a decrease in the natural yeast
population of wheat phyllosphere caused by tebuconazole and tetraconazole; such
decrease was significantly higher than that caused by azoxystrobin or sulphur (De
Curtis et al. 2012).
Concerning the epiphytic bacteria, different researchers reported that the fun-
gicides prochloraz, triadimenol, maneb, mancozeb, and triadimefon had no nega-
tive effect on the bacterial population of wheat phyllosphere (Dik and van Pelt
1992; Southwell et  al. 1999). By contrast, Walter et  al. (2007) showed that the
majority of tested fungicides negatively affected the natural bacterial population
on apple surfaces.
70 Advances in Postharvest Fruit and Vegetable Technology

4.5 INTEGRATION OF ANTAGONIST MICROORGANISMS

WITH OTHER CONTROL SYSTEMS
BCAs, when applied as stand-alone treatments under commercial conditions, rarely
exhibited high efficacy and consistency against postharvest diseases. The low persis-
tence, a narrow spectrum of activity and a failure to control previously established
infections (e.g., latent, quiescent, and incipient infections) are considered the main
limiting factors (Droby et al. 2009). Thus, it is generally accepted that a combina-
tion of BCAs with other methods is necessary to improve their efficacy (Ippolito
and Nigro 2000; Lima et al. 2006; Droby et al. 2009). Such an integrated approach,
due to additive or synergistic effects of combined or sequenced treatments, can
even provide rates of disease control comparable to or better than synthetic fungi-
cides applied alone at label dosages. Therefore, integrating BCAs with other control
means can increase their acceptance by packing houses, retailers, and consumers.
Integrative strategies include their combination with physical means, natural-derived
compounds, or even low dosage of synthetic fungicides (Lima and De Cicco 2006;
Feliziani and Romanazzi 2013). One of the most effective strategies to optimize the
efficacy of BCAs and control latent infections is the application of antagonists in
preharvest situations (Ippolito and Nigro 2000).

4.5.1 BCAs and Physical Means

The integration of BCAs with physical means such as high/low temperature, UV-C
light, ozone, and modified or controlled atmosphere has received increasing atten-
tion, since, presumably, the control effect is due not only to direct inhibition of the
pathogen but also to the induction of resistance in fruit (Droby et al. 2002). Although
cold storage retards spoilage of many produce, it is often not sufficient to fully inhibit
the postharvest pathogens throughout the required storage period. Morales et  al.
(2008) reported that two BCAs (C. sake CPA-2 and P. agglomerans CPA-1) were
able to control P. expansum growth and patulin accumulation in cold-stored (1°C)
apples. In particular, C. sake was more effective on fungal growth, whereas P. agglo-
merans reduced toxin accumulation. However, both BCAs could not control blue rot
and patulin accumulation during storage at 20°C; in some cases, they even increased
P. expansum aggressiveness.
Use of high temperature can contribute to biocontrol success. Heat (38–60°C)
may be applied to fruit and vegetable by hot-water dips (HW), steam, hot dry air, hot-
water rinsing and brushing (HWRB). For example, D’Hallewin et al. (1998) reported
that the combination of a heat treatment (37°C, 95% relative humidity (RH) for 72 h)
with C. famata produced a synergistic effect against P. digitatum on grapefruit com-
parable to that of the fungicide imazalil, when the heat treatment was performed
within 36 h after inoculation with the antagonist. Whereas, Casals et al. (2010) tested
HW (60°C for 40 s) with B. subtilis CPA-8, separately or in combination, against
Monilinia spp. infections during the postharvest storage of stone fruit. When HW
treatment was followed by CPA-8 application, a significant additive control effect
on Monilinia laxa was detected. Similarly, Massignan et al. (2005) observed a sig-
nificant reduction of green/blue mold only on oranges treated by HW (57°C, 1 min)
Biological Control of Postharvest Diseases 71

in combination with the antagonist A. pullulans, L47, as compared to the control or

HW and antagonist alone, but also found the highest reduction on fruit held at 95%
RH as compared to fruit stored at the same temperature (7°C), but at 85% RH dur-
ing storage and shelf-life. Also, treatment by B. amyloliquefaciens combined with
2% sodium bicarbonate and HW (45°C for 2 min) was as effective as the fungicide
treatment and reduced postharvest decay of mandarin fruit by more than 80% as
compared to the control. This combination significantly reduced postharvest decay
without impairing fruit quality after storage at 25°C for four weeks or at 6°C for
eight weeks (Hong et al. 2014). Promising results were obtained even with HWRB
where the fresh product is rinsed with pressurized hot water for no longer than 30 s at
48–60°C by nozzles while rolling on brushes. Treating stonefruit by HWRB at 60°C
for 20 s and then dipping into a cell suspension of the yeast Candida sp. 24 h after
inoculation with P. expansum reduced decay development by 60% compared with
the control (Karabulut et al. 2002).
Controlled atmosphere (CA) or modified atmosphere (MA) storage can delay the
onset of ripening and senescence and hence postpone the time at which produce
become more susceptible to decay, thus enhancing the biocontrol ability of antag-
onists. For example, yeasts Trichosporon sp. and C. albidus were more effective
against B. cinerea and P. expansum on apples and pears under CA (3% O2 and 3%
CO2 or 3% O2 and 8% CO2) conditions than in air (Tian et al. 2002). C. oleophila
strain O in combination with 2% calcium chloride and modified atmosphere packag-
ing (MAP) in nonperforated polyethylene bags, reduced by 53% crown rot of banana
caused by Colletotrichum musae, exerting a synergistic effect as compared to the
single treatment (Bastiaanse et al. 2010).
Since ozone treatment, in many instances, significantly reduces populations of
filamentous fungi, yeasts, and bacteria after 10 min exposure, it could negatively
affect BCAs applied to the fruit surface. However, the application of Muscodor
albus, which produces inhibitory volatiles, reduced decay incidence on grape berries
artificially infected with B. cinerea from 92% (control) to 21%. The ozone treatment
alone or in combination with M. albus reduced incidence of decay to 19% or 10%,
respectively (Mlikota Gabler et al. 2010). On organically grown grapes, where the
natural incidence of grey mold was 31%, treatment with a combination of M. albus
and ozone reduced decay incidence after one month of storage at 0.5°C to 3%, a
greater reduction than either treatment alone. Ozone treatment also reduced decay on
berries inoculated with R. stolonifer before or after treatment, indicating a possible
resistance induction.
Among eradicative treatments, 2450 MHz microwave (used in kitchen-type
microwave ovens) was successfully used to control postharvest decay, although it
had no residual protection (Karabulut and Baykal 2002). Zhang et al. (2006) reported
the effect of microwave and C. laurentii, singly and in combination, on pears. The
incidence of blue mold by P. expansum was reduced from 100% in the control to
73% after microwave treatment, to 66% using the antagonist only, and to 20% after
application of both treatments. Microwaves proved not to impair major fruit-quality
indices.
Recently, UV-C irradiation and the yeast antagonist Candida guilliermondii were
used against artificial and natural infection by P. expansum and B. cinerea in pear
72 Advances in Postharvest Fruit and Vegetable Technology

fruit stored at 20°C (Xu and Du 2012). Applied separately, both C. guilliermondii
and UV-C (5 kJ/m2) effectively inhibited rots; however, their combination showed
better control efficacy. Application of UV-C proved to not affect the BCA growth in
pear fruit wounds, while it induced a significant increase in the activities of chitin-
ase, β-1,3-glucanase, catalase, and peroxidase in fruit. The elicitation of the defense
responses in pear fruit might be accountable for the enhanced C. guilliermondii
biocontrol efficacy.

4.5.2 BCAs and Natural Compounds

Natural compounds include a wide range of products of animal, microbial, plant, or
mineral origin, many of which are included in the GRAS (Generally Recognized as
Safe) list. Several of these compounds, characterized by low toxicity toward mam-
malian and environment, display antimicrobial activity. Moreover, many are clas-
sified as food-grade additives and thus particularly suitable for combination with
BCAs (Lima et al. 2005; Sanzani and Ippolito 2011).
A successful example of a combination of an antagonist with a compound of
microbial origin is that of xanthan gum (a polysaccharide secreted by the bacterium
Xanthomonas campestris) with the yeast-like fungus A. pullulans to control posthar-
vest table grape and strawberry rots. On both commodities, the activity of the antago-
nist was significantly improved when applied in combination with the polysaccharide
at 0.5% (w/v) (Ippolito et al. 1997). The higher activity of A. pullulans combined with
xanthan gum was related to its greater survival on the fruit surface. Single applica-
tions of ethanol (8%–20%) and S. cerevisiae were not effective in reducing grey mold
on apples (Mari and Carati 1997), but their combination reduced the incidence of
disease by over 90%. Similar results were obtained against lemon green mold by
combining ethanol with C. oleophila. Infections were reduced from 82% (control) to
17% (ethanol alone), 40% (yeast alone), and 3.3% (ethanol-yeast) with no appreciable
differences compared with the fungicide imazalil (Lanza et al. 1997).
Within the plant-origin category, essential oils can be included. The combination
of B. amyloliquefaciens, strain PPCB004, with thyme (TO) and lemongrass (LO)
oils was evaluated against B. cinerea, P. expansum, and R. stolonifer on peach fruit
(Arrebola et al. 2010). The biofilm formation of PPCB004 was significantly higher
in LO than TO. LO and the BCA completely inhibited pathogen mycelial growth.
Fruit inoculation trials with PPCB004+LO in NatureFlex™ Modified Atmosphere
Packaging (MAP) showed lower disease incidence and severity at 25°C for five days
than other treatment combinations or stand-alone MAP. In particular, the combina-
tion of PPCB004+LO in NatureFlex™ MAP showed the absence of disease and off-
flavor development, retained the overall appearance and increased the acceptance at
market-shelf conditions after cold storage at 4°C for 14 days.
The effects of the plant product methyl jasmonate (MeJA) and the yeast C. lau-
rentii, alone or in combination against postharvest diseases in peach fruit, and the
possible mechanisms involved, were investigated by Yao and Tian (2005). MeJA
enhanced the population of C. laurentii and inhibited mycelial growth of P. expan-
sum. The MeJA and C. laurentii combination induced higher activities of chitinase,
Biological Control of Postharvest Diseases 73

β-1,3-glucanase, phenylalanine ammonia-lyase, and peroxidase than applying the

yeast or MAJA alone and reduced the lesion-diameter of brown rot and blue mold
caused by M. fructicola and P. expansum.
Good results were obtained combining compounds of animal origin such as
chitosan, a polysaccharide obtained from crustacean shells, with several antago-
nists. A combination of chitosan and the antagonist C. utilis proved to be effective
in controlling postharvest pathogens on tomato (Sharma et al. 2006). Table grape
bunches sprayed 10 days before harvest with the combination of 0.1% chitosan and
C. laurentii and stored for 42 days at 0°C, followed by a three day-shelf life, had a
decay index of grey mold of 0.15 (based on a 0–1 scale) compared to 0.30 recorded
in the control. Bunches treated preharvest with the same antagonist, dipped in 1%
chitosan solution after harvest and stored in the above conditions, had a decay index
of 0.35 in the control and 0.15 in treated bunches (Meng et al. 2010). Among grapes
stored at 2°C for 15 days, the decayed berries were reduced from 22% in the con-
trol to 10% by using the combined treatment of two yeasts (Pichia anomala and
Cryptococcus humicolus) with potassium caseinate and calcium chloride (Ligorio
et al. 2007).
Mineral-derived compounds are more extensively used in combined treatment
studies. For instance, A. pullulans, in combination with calcium chloride or sodium
bicarbonate (SBC), was found to be effective against postharvest pathogens on sweet
cherries (Ippolito et al. 2005). Moreover, calcium chloride infiltrations combined with
antagonist application on apples increased control of P. expansum upto six months of
storage at 1°C, compared to biological treatment alone (Janisiewicz et al. 1998).
The activity of 2 min dips in 3% sodium carbonate or sodium bicarbonate aque-
ous solutions heated to 40°C, alone or followed by the application of P. agglomerans
CPA-2 (BA) were simultaneously evaluated against citrus green mold. The combi-
nation of the salts and BCA gave the best activity in preexisting wounds, but not in
new wounds (Usall et al. 2008). In addition, Spadaro et al. (2002) found that heat
treatment (HT) and SBC significantly improved the efficacy of the BCA M. pulcher-
rima against blue mold. Similarly, enhancement of biocontrol activity was achieved
in pome fruits combining l-serine, l-aspartic acid, or ammonium molybdate with
C. sake (Nunes et al. 2001), in papaya SBC with C. oleophila (Gamagae et al. 2004).
Twelve compounds (organic and inorganic calcium salts, natural gums, and antioxi-
dants) currently used as food additives, in combination with three BCAs (R. glutinis,
C. laurentii, and A. pullulans), dramatically improved the antagonistic activity of
one or more of the tested BCA against P. expansum on apples, with additive or syn-
ergistic effects (Lima et al. 2005).
The efficacy of antagonistic yeast C. oleophila strain O, 2% calcium chloride,
and MAP in nonperforated polyethylene bags, applied alone or in various combina-
tions, was evaluated by Bastiaanse et al. (2010) under conditions highly conducive to
the development of crown rot on banana fruit artificially inoculated with C. musae.
Both antagonistic yeast and storage under MAP, applied separately, reduced crown
rot significantly whereas calcium chloride had no effect on C. musae. The yeast
showed a 16% higher biocontrol activity when applied with calcium chloride, achiev-
ing the same protective effect with a lower yeast concentration. However, the highest
74 Advances in Postharvest Fruit and Vegetable Technology

synergistic efficacy (53%) was achieved combining the three alternative control
means. A synergistic effect of the BCAs C. laurentii and R. glutinis applied in com-
bination with silicon (Si, 2%) against A. alternata and P. expansum was observed in
jujube fruit stored at 20°C but not at 0°C (Tian et al. 2005). Similarly, in a study by
Droby et al. (2003), 2% SBC enhanced the performance of the yeast-based bioproduct
Aspire (curative and protective effect) against Botrytis and Penicillium rot in apple
and Monilinia and Rhizopus rot in peach. The result of this integrated approach is
the development of a second generation of products such as “Biocoat,” whose main
components are C. saitoana and chitosan, or “Biocure” with C. saitoana and lyso-
zyme (Micro Flo, Memphis, TN, USA). Both products contain other additives such
as sodium bicarbonate (SBC) (Wisniewski et al. 2007). The bioactive coating activ-
ity was found to be superior to each component in controlling decay of several variet-
ies of sweet orange, lemons, and apples, with a control level comparable to that of the
fungicides imazalil and thiabendazole (El Ghaouth et al. 2000; Schena et al. 2005).

4.5.3 BCAs and Low Dosage of Synthetic Fungicides

Most BCAs show good resistance in vitro to fungicides (e.g., anilides, anilinopy-
rimidines, benzimidazoles, chlorothalonil, copper salts, dicarboxymides, dithio-
carbamates, strobilurines, sulphur, and triazoles) commonly applied on fruit and
vegetables in the field or postharvest (Omar et al. 2006; Lima et al. 2008). Fungicides
based on copper or sulphur (which are also allowed in organic agriculture), in several
cases showed a slight effect on yeasts and yeast-like fungi, whereas products based
on synthetic chemical fungicides have an activity that depends on the species of the
BCA and the chemical group to which the fungicide belongs.
In specific studies, BCAs compatible with low doses of chemical fungicides were
evaluated. Lima et al. (2003) observed that some strains of C. laurentii and A. pul-
lulans were more resistant to high concentrations of benzimidazoles and dicarboxi-
mides than strains of R. glutinis, while the sensitivity to triazole fungicides was high
and similar for all three BCAs.
Compatibility with chemicals is an important trait for a more appropriate utiliza-
tion of a BCA in an integrated control schedule. An antagonist can interact with the
pathogen and also with fungicides applied before or after harvesting (Lima et  al.
1997; Buck and Burpee 2002; Ippolito et  al. 2004). Therefore, the optimization
of antagonist-efficacy depends on its survival and colonization of unwounded and
wounded plant surfaces in the presence of fungicides. Numerous studies performed
in commercial packing houses demonstrated that integrating BCAs or their based
commercial formulates with small quantities of synthetic fungicides showed higher
efficacy and persistence against postharvest decay of several important fruit, some-
times displaying an efficacy comparable to the fungicide applied alone at the full
label rate (Lima et al. 2003). Furthermore, studies have also shown that a combina-
tion of biocontrol yeasts with small quantities of synthetic fungicides exerted a more
efficient control of both fungicide-sensitive and fungicide-resistant strains of fungal
pathogens (Lima et al. 2006) and reduced fungicide residues and accumulation of
mycotoxins in fruit (Lima et al. 2011).
Biological Control of Postharvest Diseases 75

4.5.4 Preharvest Application of BCAs

Postharvest quality of fresh fruit and vegetables depends on the quality of pro-
duce at harvest. Important components of the preharvest environment include
plant pathogens; indeed, produce that appears healthy at harvest may harbor
latent, quiescent, or incipient infections capable of causing significant losses dur-
ing storage. Their impact has generally been underestimated due to postharvest
application of fungicides with a strong curative activity. In addition, many fun-
gicides applied just before harvest can control postharvest diseases (Feliziani
and Romanazzi 2013). However, as stated above, nowadays their use has been
strongly restricted, the time interval between application and harvest has been
increased, and residues are no longer accepted. Available alternative control
measures, including BCAs, fail to control infections initiated before treatment
(Sanzani et  al. 2009). Latent and quiescent infections in fruit and vegetables
(Snowdon 1990; Sanzani et al. 2012) in some instances can prevail over infec-
tions occurring during and after harvest (Ippolito and Nigro 2000). Based on
this consideration, the best system to control preharvest infection is prevention,
including application of BCAs before harvest. There are few published examples
of preharvest applications of BCAs but there is an increasing interest towards
preharvest treatments to reduce field latent/quiescent infections or induce fruit
resistance as part of an integrated diseases management program (Feliziani
et al. 2013). It was shown that antagonists preemptively colonized flower parts
to an extent that their activity prevented the colonization of senescent stamens
by B. cinerea (Lima et al. 1997). The application of A. pullulans and Epicoccum
purpurascens to sweet cherry blossoms reduced the number of latent infections
by M. laxa in green fruits (Wittig et  al. 1997). Also, in case of stem-end rot
of avocado, the application of B. subtilis strain B246 at flowering was effec-
tive in reducing the incidence of disease (Korsten et  al. 1997); the antagonist
extensively and steadily colonized flower parts, multiplying in specific niches
and preventing pathogen establishment and germination (Demoz and Korsten
2006). These findings suggest that the application of BCAs at flowering stage is
a good control strategy when the pathogen gains entrance through flower parts.
However, depending on the epidemiology of the disease, other moments of pre-
harvest application can be effective. In apples, late application (August) of a
mixture of antagonists was found to effectively suppress postharvest rots, mainly
those due to Pezicula spp., Penicillium spp., and M. fructigena (Leibinger et al.
1997). Four strain of Bacillus spp. applied in May, or May and June, significantly
reduced fruit and foliar apple-scab severity (Poleatewich et al. 2012), while the
May + June + postharvest application of Bacillus megaterium, and isolate A3-6,
resulted in the greatest suppression of bitter rot by C. acutatum, with an aver-
age of 45% and 95% reduction in the lesion size compared to nontreated apples
(Poleatewich et al. 2012). On sweet cherries, A. pullulans and salts applied alone
and in combination, one week before harvest, were as effective as the fungicide
tebuconazole in controlling postharvest rots (Ippolito et al. 2005).
Other benefits in applying antifungal preharvest treatments are to reduce field
populations of the pathogen, suppress the pathogen at the source, and induce
76 Advances in Postharvest Fruit and Vegetable Technology

resistance in the fruit (Palou et  al. 2008). Regarding the first aspect, Lima et  al.
(1997) observed a significant decrease of filamentous fungi population including B.
cinerea, only on A. pullulans L47 treated strawberries but not in those fruit treated
with a weak antagonist (C. oleophila, strain L66). Regarding the second aspect, an
example of the induction of resistance is the combination of preharvest application
with C. laurentii and postharvest chitosan coating, which was found to significantly
decrease table grape decay index; the results were ascribed to the strongest increase
of activity of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) in
combined treatments (Meng et al. 2010).
The preharvest application of BCAs seems an appropriate strategy also, where
postharvest handling is unacceptable because the produce may appear less appeal-
ing. This relates to fruit that are easily damaged or cannot be exposed to water-
based treatment such as strawberries, and fruit with a waxy bloom on the surface
such as table grapes. Postharvest treatment with P. guilliermondii or Hanseniaspora
uvarum was found to markedly suppress the postharvest decay of table grapes; but
it also tended to remove the surface bloom. This problem was avoided by applying
P. guilliermondii three days before harvest (Ippolito and Nigro 2000). In various
parts of the world, to avoid handling, table grape bunches are directly packed in the
field and stored at low temperature. In this situation, only fumigation with gaseous
substances such as sulfur dioxide or ozone is feasible. For similar reasons, it is
preferable to harvest soft fruit and pack them directly into plastic baskets for mar-
keting through to consumer. In these situations, spraying suspensions of antagonist
propagules one day before harvest, using the same equipment as for pesticide appli-
cations, is feasible.
Knowledge of the epidemiology of the target disease is crucial for choosing the
right time for the application of BCAs. Under commercial conditions, the antago-
nist may encounter wounds colonized by resident microbial flora. In such situ-
ations, a preharvest or near-harvest application of the antagonist could permit a
preemptive colonization of the wound immediately after it has been inflicted, satu-
rating the fresh wound before the arrival of the pathogen (Janisiewicz and Korsten
2002). Competitive or preemptive exclusion is considered to be the primary mech-
anism by which pear and apple blossoms are protected against fire blight by apply-
ing E. herbicola or P. fluorescens (Stockwell et al. 1998). On strawberries treated
with A. pullulans immediately before harvest, Rhizopus rot was reduced by 72%
although only slight activity was observed against Botrytis rot (Lima et al. 1997).
This difference can be explained on the basis of a different disease cycle of the
pathogens: B. cinerea was present inside the fruit as a latent infection, while R. sto-
lonifer was an external contaminating pathogen infecting ripe strawberry through
wounds on the surface.
Finally, application of BCAs before harvest implies that the antagonist should
have the capability to survive at a high population rate, despite difficulties encoun-
tered in the field environment. Some BCAs, such as A. pullulans, have these charac-
teristics (Ippolito and Nigro 2000). In other cases, environmental stress tolerance has
been enhanced, gaining good rot control performance, as for C. sake strain CPA-1
(Teixidó et al. 2010).
Biological Control of Postharvest Diseases 77

4.5.5 Multifaceted Approaches
A multifaceted approach can be defined as the combined or sequenced integration
of the most common and available field and postharvest control methods (e.g., agro-
nomic, biological, chemical, genetic, and physical). According to the integrated pest
management system, this multistrategy approach, if mainly addressed to a preven-
tive control of postharvest diseases, can maximize the efficacy of BCAs by reducing,
to the lowest possible level, the use of synthetic fungicides. As also evidenced by
various examples of combined treatment reported in other sections of this chapter,
multifaceted integrated approaches can provide additive or synergistic activity to
biocontrol treatment, so that the BCA can completely control the development of
postharvest decay (Lima and De Cicco 2006; Palou et al. 2008; Janisiewicz 2013).

4.6  BCA FORMULATION

Formulation of microorganisms for biocontrol of plant pathogens is undeveloped, if
compared to other applications of microorganisms (Janisiewicz and Korsten 2002).
However, considering that most of the positive results on biological control of post-
harvest diseases of fruit and vegetables were obtained by applying microbial antag-
onists as acqueous cell suspension, the potential to set, up efficient formulates of
microbial antagonist is very high.
Data on the formulation of the most promising BCAs is often not accessible to
the public, since it is obtained by a private company and seldom published. However,
as evidenced in other sections of this chapter, research activity aimed at enhancing
antagonist viability, efficacy and shelf-life is increasing and the presence of spe-
cific compounds seems to be an essential prerequisite for the commercial success of
antagonist-based biofungicides. Adjuvants are compounds of various origins which,
generally, if used alone at a concentration compatible with fruit, do not exert any
direct activity against pathogens. However, they can exert additive or synergistic
effects on biocontrols, if combined with BCAs. Several natural molecules such as
food-grade additives, edible coatings, plant or animal extracts, enzymes or antioxi-
dants were found to improve biocontrol efficacy against a range of postharvest patho-
gens (see also Section 4.5.2).
Among these compounds, antioxidants are of particular interest. In fact, the dem-
onstration of the role of resistance to oxidative stress, as a key mechanism in the bio-
control exerted by antagonistic yeasts against postharvest wound pathogens, paves
the way to the improvement of the activity of BCAs in wounds by combining with
antioxidants (Castoria et al. 2003).
Manipulation and storage of microbial antagonist formulates are more prob-
lematic than formulates of chemical pesticides. Performance and stability of BCA-
based products can be greatly affected by a variety of factors, such as water, food,
and environment. Another crucial factor for some antagonists is the high cost of
their biomass production. However, when applied in combination with effective
adjuvants and/or low doses of fungicides, a lower BCA cell concentration can be
required to obtain high antagonistic efficacy. This could, in turn, reduce the cost
78 Advances in Postharvest Fruit and Vegetable Technology

of fermentation for biomass production, making more realistic the potential for
developing new and highly effective BCA formulations for large-scale applications
against postharvest diseases.

4.7 GENOMIC APPROACHES TO STUDY BCA–

HOST–PATHOGEN INTERACTIONS
In recent years, advanced molecular techniques greatly contributed to improv-
ing knowledge on the mode of action of BCAs and providing insights into their
detection and population dynamics. Indeed, conventional detection methods often
do not enable the identification of specific strains and the population of BCAs can
be influenced by several factors, including time and mode of application, capabil-
ity for colonization, survival in unfavorable conditions, and tolerance to chemical
treatments (Sanzani et al. 2014). Furthermore, a prerequisite for the registration of
effective BCAs is the assessment of environmental risks related to their distribution,
since they should not have nontarget effects on the environment and/or nontarget
organisms (Gullino et al. 1995). A decisive boost was given by the invention in 1984
of polymerase chain reaction (PCR) by Kary Mullis. For example, the repetitive
sequence-based PCR (rep-PCR) uses primers targeting several of these repetitive
elements to generate DNA profiles or “fingerprints” of individual microbial strains
(Ishii and Sadowsky 2009). In screening strategies, these fingerprints can be applied
to differentiate strains at population level and to select unique isolates; in in vivo
assays, they can be used for identity and quality control purposes (Berg et al. 2006).
Random amplified polymorphic DNA (RAPD) markers proved also to be useful in
estimating genetic variations; for instance, they were applied to obtain fingerprint-
ing patterns among 16 strains of Trichoderma asperellum, T. atroviride, T. harzia-
num, T. inhamatum, and T. longibrachiatum previously selected as BCAs (Hermosa
et al. 2001). The obtained SCAR (sequence-characterized amplified region) marker
clearly distinguished T. atroviride strain 11 from other closely related Trichoderma
strains. Finally, the usefulness of the amplified fragment-length polymorphism
(AFLP) technique for the genetic analysis of 26 M. pulcherrima strains, isolated
from different sources in different geographical regions, was confirmed by Spadaro
et al. (2008). Genetic relationships between strains were also estimated using AFLP:
all the isolates, previously tested as BCAs, were grouped in a single cluster with a
high bootstrap value indicating robustness and reproducibility. Fluorescent AFLP
(fAFLP) analysis was used to investigate the intraspecific variability of the yeast-like
fungus A. pullulans strain LS30, in order to identify specific molecular markers for
tracking this agent in the environment (De Curtis et al. 2004). Forty-eight isolates of
A. pullulans from phyllosphere and carposphere of several crops from different sites
of Greece and southern Italy were analyzed. Most of the isolates grouped into three
main clusters, but only two (AU73 and AU91) were very similar in all fAFLP pat-
terns. Three DNA fragments that appeared to be specific for strain LS30 were found.
However, the potential of conventional PCR was greatly increased by the develop-
ment of real-time quantitative amplification technologies (qPCR) (Schena et al. 2013).
For example, qPCR methods based on the use of SCAR regions have been utilized
to differentiate field-applied biocontrol strains from autochthonous wild populations
Biological Control of Postharvest Diseases 79

of the same species or genus (Sanzani et al. 2014). A strain of A. ­pullulans (L47),
was monitored and quantified on the carposphere of table grapes and sweet cherries,
demonstrating that its population increased soon after application and remained high
over the growing season (Schena et al. 2002). Vallance et al. (2009) studied the influ-
ence of the BCA Pythium oligandrum on fungal and oomycete population dynamics
of the rhizosphere and found that, with few exceptions, there were no significant
differences between the microbial ecosystems inoculated with P. oligandrum and
untreated systems.
A recent evolution of qPCR, High Resolution Melting (HRM), based on the anal-
ysis of the melt peak, has significant potential to enhance the currently available
detection protocols. Indeed, different regions within a PCR amplicon sometimes
denature from double-stranded to single-stranded at different temperatures because
of varying thermal stabilities between regions. This results in a unique melt profile
for the amplicon. HRM application for a simple and efficient detection of BCAs has
been recently reviewed by Monk et al. (2011).
Genome sequencing also offers a tool to study BCAs in great detail. Strains of
P. fluorescens were the first to be sequenced (Paulsen et al. 2005). Indeed, genomic
information allows the analysis of the mode of action and interactions, as well as the
optimization of formulation processes (Gross and Loper 2009). Moreover, as a con-
sequence of genomic information availability, gene inactivation and over-expression
studies can be performed, providing information on the transcription and regulation
of these genes. De Bruijn et al. (2007) used genome mining to discover unknown
gene clusters and traits highly relevant to P. fluorescens SBW25. Catalano et  al.
(2011) used gene deletion for studying the involvement of laccases in the degrada-
tion of sclerotia of plant pathogenic fungi by the mycoparasitic fungus T. virens. The
laccase gene lcc1, expressed after the interaction with sclerotia of the plant patho-
genic fungi B. cinerea and Sclerotinia sclerotiorum, was deleted, obtaining a mutant
altered in its ability to degrade the sclerotia. Interestingly, while the decaying ability
for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum
sclerotia was enhanced, suggesting different mechanisms in the mycoparasitism of
these two sclerotia types by lcc1.
Biocontrol activity could even involve reduction of mycotoxin levels. Mycotoxins
are not only a safety issue but may be involved in the pathogen virulence/pathoge-
nicity (Sanzani et  al. 2012). Molecular tools can also be useful in evaluating the
detoxifying activity of BCAs. For instance, Sporobolomyces sp. IAM 13481 and
R. kratochvilovae strains were found to be able to degrade patulin to less toxic
breakdown products, desoxypatulinic acid and (Z)-ascladiol (Castoria et al. 2011;
Ianiri et al. 2013). To gain insight into the genetic basis of tolerance and degrada-
tion of patulin, mutants were generated and screened. The patulin-sensitive mutants
also exhibited hypersensitivity to ROS, and genotoxic and cell-wall-destabilizing
agents, suggesting that inactivated genes are essential for overcoming the toxicity
of patulin.
Transcriptomic studies are particularly useful to study the function of BCAs.
For example, Garbeva et  al. (2011), studying the transcriptional and antagonistic
responses of P. fluorescens Pf0-1 to phylogenetically different bacterial competitors,
demonstrated the existence of a species-specific response. Perazzolli et  al. (2011)
80 Advances in Postharvest Fruit and Vegetable Technology

showed that T. harzianum T39 reduced downy mildew severity on susceptible grape-
vines under controlled greenhouse conditions by a direct modulation of defense-
related genes and the activation of priming for enhanced expression of these genes
after pathogen inoculation. Transcriptomic studies can also lead to new insights into
plant responses on BCAs: Pseudomonas-primed barley genes indicated that jasmonic
acid plays a role in host responses (Petti et al. 2010).
Microarray-based experiments have focused on model organisms whose genomes
have been completely sequenced. Hassan et  al. (2010) developed a whole genome
oligonucleotide microarray for P. fluorescens Pf-5 to assess the consequences of
a gacA mutation; gadA significantly influenced transcript levels of genes involved
in the production of hydrogen cyanide, pyoluteorin and the extracellular protease.
Similarly, microarrays were used to gain insight into the mechanism by which
Trichoderma hamatum 382 induced resistance in tomato, high-density oligonucle-
otide (Alfano et al. 2007). It proved to have the ability to consistently modulate the
expression in tomato leaves of genes associated to biotic or abiotic stress, as well as
RNA, DNA, and protein metabolism.
Methods for the in situ analysis of antifungal gene expression using green fluores-
cent protein (GFP)-based reporter fusions have been established. Marker genes were
introduced in the biocontrol strain Clonostachys rosea IK726 as a tool for monitor-
ing the strain in ecological studies (Lübeck et al. 2002). The β-glucuronidase (GUS)
reporter gene and a green fluorescent protein (GFP) encoding gene were, in separate
experiments, integrated into the genome of IK726. Compared to the wild type, the
two selected GUS and GFP transformants maintained the ability to colonize barley
roots, and to efficiently reduce the severity of Fusarium culmorum without affect-
ing plant emergence. Nigro et  al. (1999) transformed a strain of M. pulcherrima
with the yeast-enhanced GFP (yEGFP). The activity of two obtained transformants
was indistinguishable from that of the parental strain, significantly reducing Botrytis
storage rot.
The innovative Next Generation Sequencing (NGS) approaches could greatly
affect the efficacy and registration of BCAs, providing a plethora of information in
a single experiment. Hershkovitz et al. (2013) performed a transcriptome analysis
using RNA-Seq technology to examine the response of the BCA M. fructicola to
citrus fruit and to the postharvest pathogen Penicillium digitatum. An analysis of
differential expression, when the yeast was interacting with the fruit vs. the patho-
gen, revealed more than 250 genes with specific expression responses. In the antago-
nist–pathogen interaction, genes related to transmembrane, multidrug transport, and
amino acid metabolism were induced. In the antagonist–fruit interaction, expression
of genes involved in oxidative stress, iron and zinc homeostasis, and lipid metabo-
lism were induced.

4.8  CONCLUDING REMARKS

The use of microbial antagonists has been intensively explored and considered over
the last three decades as a promising method to control postharvest diseases as an
alternative to the use of synthetic fungicides. However, despite the extensive research,
very few BCA-based products are commercially available for use against postharvest
Biological Control of Postharvest Diseases 81

pathogens. This contrasts with the high number of biopesticides registered for field
application against rhizosphere or phyllosphere pathogens.
Various obstacles and constraints limiting the commercial implementation of
BCAs in the control of postharvest diseases have been identified by researchers and
fruit-industry operators. However, the most recent research on biological control of
postharvest diseases has given various suggestions to overcome most of the limiting
factors, raising the potential of BCAs as a valuable alternative to synthetic pesticides.
For a satisfactory biocontrol of a postharvest plant pathogen, it is of paramount
importance to determine the most appropriate strategy of application according
to the disease cycle. Further studies on microbial ecology of plant surfaces in the
field can also contribute to improve survival, colonization, and preventive activity
of microbial antagonists. Another crucial aspect to enlarge the market potential of
biocontrol products is the integration of antagonists with various control means,
including natural compounds as well as genetic, agronomic, chemical and/or physi-
cal tools. Moreover, to optimize fruit protection and quality, the global chain of
fruit and vegetables production and manipulation must be considered by combining
preharvest and postharvest interventions.
On the improvement of effectiveness and shelf-life of BCA formulations, consid-
erable progress is expected in the coming years by adding biocontrol formulations
with suitable new adjuvants that can markedly reduce the amount of microbial bio-
mass in BCA formulates without reducing biocontrol efficacy.
In integrated agriculture systems, combining low dosages of fungicide with com-
patible antagonists seems one of the most immediate possibilities for large-scale
use of BCAs by growers and packing houses. Such integration not only consistently
reduces the amount of fungicides needed and the related risks, but can also con-
trol both sensitive and resistant isolates of fungal pathogens. In organic agriculture
systems of horticultural commodities to be exported to markets that require zero
pesticide residues and organically labeled fruit, the integration of BCAs with other
nonchemical alternatives can allow a satisfactory control of postharvest rots.
In conclusion, a great deal of research has shown that using antagonistic micro-
organisms in integrated approaches can lead to results comparable to or even better
than chemicals, thus increasing interest for implementation and large-scale appli-
cations of BCA-based products and stimulating the registration of new antagonist-
based formulates.

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 5 Physical and
Chemical Control of
Postharvest Diseases
Alice Spadoni, Fiorella Neri, and Marta Mari

CONTENTS
5.1 Introduction..................................................................................................... 89
5.2 Physical Control of Postharvest Diseases........................................................90
5.2.1 Heat Treatments...................................................................................90
5.2.2 Irradiation............................................................................................ 93
5.3 Chemical Control of Postharvest Diseases...................................................... 94
5.3.1 Generally Regarded as Safe................................................................. 94
5.3.2 Electrolyzed Water...............................................................................96
5.4 Natural Compounds......................................................................................... 96
5.4.1 Volatile Organic Compounds from Plants...........................................96
5.4.2 Isothiocyanates....................................................................................97
5.4.3 trans-2-Hexenal................................................................................... 98
5.4.4 Carvacrol and Thymol.........................................................................99
5.4.5 Citral.................................................................................................. 100
5.4.6 trans-Cinnamaldehyde...................................................................... 100
5.4.7 Essential Oils..................................................................................... 101
5.4.8 Volatile Organic Compounds from Microorganisms........................ 102
5.4.9 Chitosan............................................................................................. 103
5.5 Compounds Inducing Resistance................................................................... 104
5.6 Integrated Methods........................................................................................ 105
5.7 Conclusions.................................................................................................... 106
References............................................................................................................... 106

5.1 INTRODUCTION
Fruit and vegetables are highly perishable produce and, after harvest, are subjected
to considerable quantitative losses. Since they are rich in water and nutrients, such
produce are ideal substrates during storage for the development of pathogenic micro-
organisms such as Penicillium spp., Monilinia spp. and Botrytis cinerea. Waste, pro-
duced during the postharvest phase, also represents an important economic loss,
considering the added value of fruit after harvest, storage, transport, and commer-
cialization. The extent of postharvest losses varies, depending on the commodities

89
90 Advances in Postharvest Fruit and Vegetable Technology

and countries in question, and although little uptodate data is available, losses can
be estimated to range from a minimum of 10%–15% in countries with advanced
technologies to over 50% in developing countries (Wilson and Wisniewski, 1989). In
addition, qualitative losses such as loss in edibility, nutritional quality, caloric value,
and consumer acceptability of produce are much more difficult to assess than quan-
titative losses (Kader, 2005). Moreover, some postharvest pathogenic fungi such as
Penicillium spp. and Aspergillus spp. represent a serious concern to human health
since they are producers of several toxic compounds (e.g., patulin, citrinin, chaeto-
globosins, ochratoxin A) that can affect the safety of both fresh fruit and fruit-based
products (Andersen et al., 2004; Guzev et al., 2008).
The natural resistance of fruit to disease declines with ripeness and storage dura-
tion, although the use of appropriate postharvest technologies has greatly reduced
losses from production to retail in the more developed countries (Kader, 2005).
Fungicide treatments still remain one of the most effective methods to reduce post-
harvest decay, since they protect the fruit from infections occurring before treat-
ment, including quiescent infections, as well as from infections during storage,
handling, and marketing (Adaskaveg and Forster, 2010). However, the repeated and
continuous use of fungicides has led to a strong selection pressure in pathogen popu-
lations, resulting in the development of resistance to some common fungicides like
benzimidazole, demethylation inhibitors, and dicarbossimide. Moreover, in the last
25 years, concern about the effect of chemicals on human health and the environ-
ment has intensified consumer demand for fruit and vegetables with low fungicide
residues and grown with sustainable agriculture, integrated crop management and
organic production (Directive2009/128/EC) practices. Multiple retailers have identi-
fied the occurrence of pesticide residues as one of the prime concerns of consumers
regarding fresh produce, leading them to pursue a policy of residue reduction to 3–4
active ingredients or to their complete elimination (Cross and Berrie, 2008). In this
context, considerable efforts are devoted by researchers to finding safer methods
for disease control. This review deals with the substantial progress achieved in the
use of physical and chemical control measures, and also considers constraints and
obstacles that make their widespread diffusion and practical application difficult.

5.2  PHYSICAL CONTROL OF POSTHARVEST DISEASES

5.2.1 Heat Treatments
Heat treatments to control postharvest disease are usually applied at temperatures
above 40°C for a relative short time (usually 55–60°C for 20–60 s, or 45–50°C for
5–20 minutes). The inactivation of fungal propagules usually increase with tempera-
ture and the duration of the treatment; however, excessive temperatures should be
avoided due to detrimental effects on produce quality (i.e., phytotoxic symptoms,
discoloration, water loss) and short treatment durations are preferable in pack-
ing houses. The choice of the most suitable heat treatment to control postharvest
pathogens is related to the thermal sensitivity of the commodity and the target
pathogen, and is also influenced by the location and form of the pathogen in or on
the host (Barkai-Golan and Phillips, 1991; Lurie, 1998; Janisiewicz and Conway,
Physical and Chemical Control of Postharvest Diseases 91

2010; Vigneault et al., 2012). Hot-water treatments were first reported in 1922 to con-
trol decay on citrus fruit (Fawcett, 1922), but extensive reviews have subsequently
dealt with the possibility of using heat to control quarantine insects, prolong the
shelf-life of fruit and vegetables, and to prevent the development of disorders trig-
gered by cold-storage, like chilling injury (Shellie and Mangan, 2000; Fallik, 2004).
Heat treatments can be applied to fruit and vegetables in the form of hot water, vapor
heat, hot air or hot water rinse-brushing. Hot-water treatment appears to be one of
the most effective and promising methods and is especially useful for organic crops
to control relatively high rates of postharvest decay in environmentally-friendly ways
(Mari et al., 2007; Karabulut et al., 2010; Liu et al., 2012, 2013). The activity of hot-
water dipping (HWD) depends on at least two components: the first is a direct and
lethal action of heat on fungal inoculum as spores and mycelium present on the fruit
surface or in the first layers of peel; the second component could be an indirect action
of heat on the host, mediated by a stress-induced response of fruit (Maxin et  al.,
2012), including resistance to disease. The efficacy of fungal eradication depends on
genetic differences among fungi. For example, M. fructicola, is more heat sensitive
than P. expansum (Barkai-Golan and Phillips, 1991) but more resistant than M. laxa
and M. fructigena (Spadoni et al., 2013). In terms of host response, several studies
have shown that heat treatments can induce a stress response in fruit (Liu et  al.,
2012); however, more investigations are required to better elucidate the molecular
mechanisms involved.
It has been reported that appropriate heat treatments can delay ripening in sev-
eral fruit species (Paull, 1990; Lurie, 1998; Martinez and Civello, 2008), resulting
in lower softening rates in fruit and was found to have extended the postharvest life
of nectarine (Fruk et al., 2012), peach (Bustamante et al., 2012), papaya (Li et al.,
2013), banana (Promyou et al., 2008), and citrus (Yun et al., 2013). Heat treatments
are also effective for better preparing peach fruit for subsequent storage (Lauxmann
et al., 2012), avoiding or reducing the effect of some abiotic or biotic stress on fruit
quality. At the molecular level, the main factor involved seems to be the expression
of heat shock genes encoding different heat shock proteins (HSPs). It is known that
HSPs perform a critical function in refolding partially denaturated protein, complet-
ing degradation of denaturated proteins, assisting the ex novo protein synthesis and
averting protein aggregation (Aghdam et al., 2013). HWD might be a good solution
to improve fruit postharvest life but some negative aspects have to be considered. As
reported by Schirra and D’hallewin (1997), prestorage dipping of “Fortune” man-
darins in water at 50–54°C significantly inhibited chilling injury but increased the
fruit’s weight loss. Moreover, while this method has the potential to reduce the natu-
ral inoculum presents on the fruit surface, it is not always compatible with fresh-cut
plant foods since it may reduce consumer acceptability due to undesirable changes of
flavor, texture, color and nutritional quality (Maghoumi et al., 2013). Although HWD
requires specialized equipment, it might result in substantial economic saving since,
in the industrial packing house, the heating of the water can be obtained by refriger-
ant gas coming from the cooling plant of the stores; indeed, the heat of this gas is
currently usually dispersed in the environment through the condensers.
A more recent technology is the hot-water rinsing and brushing (HWRB). HWRB
treatments are extensively studied because they require a higher temperature and a
92 Advances in Postharvest Fruit and Vegetable Technology

shorter exposure time than traditional hot-water dips. HWRB treatments could not
only remove the heavy dirt, pesticides and fungal spores on freshly harvested pro-
duce but they could also improve general produce appearance and maintain produce
quality (Fallik, 2004). The method involves the fruit rolling over brushes directly
into the pressurized recycled hot-water rinse at temperatures between 48°C and 63°C
for 10–20 s (Fallik, 2004). HWRB treatments possess the ability to remove fungal
pathogens from the fruit surface as a result of the brushing effect, while the natural
wax platelets could be melted and smoothed to seal stomata openings on the fruit
surface (Fallik, 2004). It has been demonstrated that the use of HWRB at 55°C
for 12 s for sweet peppers is a practical strategy for reducing weight loss, chilling
injury and softening and preserving nutritional quality, especially antioxidant activ-
ity (Ilić et  al., 2012). However, this methodology did not have such good results
as HWD when applied to apple. In fact, the main advantage of hot-water immer-
sion is a uniformly consistent temperature profile throughout the treatment tank at
or slightly above the set point temperature, while in the case of HWRB a possible
nonuniform coverage of fruit by hot water could result in limited pathogen control.
However, HWRB has the potential to become a sustainable alternative for disease
control in fruit because it is less costly than HWD and because its shorter treatment
time enables it to be integrated into existing fruit-grading (Maxin et al., 2012).
Heat can also be applied for an extended period of time at lower temperatures.
Hot-air treatment, called “curing,” consists of holding the fruit at 30 to 37°C and at
high relative humidity (90%–98%) for 65–72 h. Forced hot air is preferred in many
countries for the development of quarantine procedures (Vigneault et  al., 2012).
However, the method based on curing treatments has also been evaluated to control
green and blue molds on citrus fruit (Zhang and Swingle, 2005; Montesinos-Herrero
et  al., 2011), proving an effective alternative to fungicides. In apple fruit, hot-air
treatment at 38°C for four days has been considered the optimum to preserve post-
harvest storage quality. It can delay ripening and maintain firmness of the apple fruit
to improve consumer acceptability, also controlling the development of common
postharvest diseases caused by P. expansum, B. cinerea, and C. acutatum (Klein
et al., 1997; Conway et al., 2005; Shao et al., 2007). As recently reported by Zhang
et al. (2013), the application of curing to tomato fruit (38°C for 12 h) activated argi-
nine catabolism and consequently reduced the susceptibility to chilling injury. In
addition, forced-hot-air treatment for prolonging postharvest shelf-life of mango did
not cause injury to the fruit but the moisture content of the heating air differen-
tially modulated the postharvest ripening; in particular, moist air temporarily slowed
down the ripening process of mangoes (Ornelas-Paz and Yahia, 2013). Curing can
also induce increased residual activity (Shao et al., 2009) and enhance the wound-
healing process (Shao et al., 2010) in controlling fungal diseases in “Fuji” apple fruit.
However, some negative impacts, including enhanced yellowing of peel, reduced
titratable acidity and weight loss were observed after heat treatment in apples. These
three factors are regarded as the signals of senescence for fruit. In a previous study,
Lurie and Klein (1992) suggested that these adverse outcomes of heat treatment could
be reduced when combined with a calcium dip. A specific curing application to kiwi-
fruits against stem-end rot caused by B. cinerea is considered an important tool in
view of a reduction in fungicide treatment. In this case, curing involves keeping fruits
Physical and Chemical Control of Postharvest Diseases 93

at ambient temperature for at least two days before cold storage (Mari et al., 2009).
During the early phases of wound-healing, active phenol metabolism was observed
in the fruits and it probably provided the monomers required for the suberization
of picking wounds, the main infection site of B. cinerea in kiwifruits. The notable
increase in phenylalanine ammonia-lyase and polyphenolxidase activity shown by
cured fruits, respectively indicated the activation of a resistance mechanism and an
oxidative phenolic polymerization, which further increased tissue resistance to the
pathogen (Ippolito et al., 1995). Currently, for grey mold management in kiwifruit,
curing is carried out directly in cold-storage rooms, decreasing the temperature from
10 to 1°C in eight days and delaying establishment of the controlled atmosphere
regimes to 30–40 days after harvesting. This method reduced the incidence of stem-
end rot but no negative effects were observed in fruit quality (Brigati et al., 2003).
Heat-based treatment has advantages over other nonconventional control methods
of fruit disease such as natural and GRAS (generally recognized as safe) compounds
(described and discussed in Section 5.3.1 below), since it does not require any regis-
tration from the European Community and ready to use. In addition, HWD or curing
appear particularly attractive to the fruit industry since it could be immediately uti-
lized and incorporated into handling practices before storage and without extensive
technical modifications.

5.2.2 Irradiation
Irradiation involves exposing the produce to controlled levels of radiation for
a specified period. The effects of irradiation on food have been extensively stud-
ied for many years (Burton et al., 1959) and are well reviewed by Arvanitoyannis
et al. (2009). Only in the last few years, due to the strong demand for reducing the
application of chemical fungicides to fruits and vegetables, investigations on the
use of different types of irradiation have been increasing and radiation technology
has now been shown to be effective in reducing postharvest losses and controlling
the stored produce against insects and microorganisms. Internationally, the World
Health Organization (WHO), the Food and Agriculture Organization (FAO), and the
International Atomic Energy Agency in Vienna (El-Samahy et al., 2000) have con-
sidered food irradiation a safe and effective technology. Among the ionizing radia-
tion technologies, gamma ray irradiation has been widely investigated on different
fruits and vegetables against postharvest fungal pathogens as B. cinerea (Jung et al.,
2014), P. expansum (Mostafavi et al., 2011) and Colletotrichum gloeosporioides (Cia
et al., 2007); these generally show a dose rate limitation to control postharvest fruit
diseases. However, depending on the irradiation dose, changes in product quality
have been observed. Doses above 2 kGy of gamma irradiation are necessary to con-
trol some postharvest pathogens, but “Fuji” apple and “Niitaka” pear irradiated at
the doses above 0.8 kGy showed an alteration in the physiological progress of firm-
ness (Jung et al., 2014). In addition, sensory qualities of irradiated grapefruit were
comparable to the untreated control fruit after 35 days’ storage only when the dose
of the gamma irradiation was 0.4 kGy (Patil et al., 2004), while the appearance of
ten citrus cultivars were negatively affected by the loss of glossiness after treatment
with 0.45 kGy (Miller et al., 2000). In order to overcome this issue, the integration of
94 Advances in Postharvest Fruit and Vegetable Technology

gamma irradiation at low doses with other treatments such as BCAs was explored,
showing a suitable method to sustain fruit quality and reduce fruit losses during stor-
age (Mostafavi et al., 2013).
The use of nonionizing radiation has also been of interest. The application of
UV-C radiation has been shown to control P. expansum of apples (de Capdeville
et al., 2002), postharvest rot of strawberries (Marquenie et al., 2002) and Rhizopus
soft rot of tomatoes (Stevens et al., 1998). The activity of UV-C, assayed in in vitro
trials, showed an inhibition of mycelial growth of Monilinia spp. after long-wave
(320–380 nm) UV exposure (De Cal and Melgarejo, 1999) but had no effect against
brown rot after UV-C light treatment was observed in cherries (Marquenie et  al.,
2002) and in peaches (Bassetto et  al., 2007). In addition, radio frequency energy
was proposed not only to heat foods but also to disinfect commodities and to control
stonefruit postharvest diseases such as brown rot (Casals et al., 2010). The effect of
UV radiation has been associated with both germicidal properties as well as physi-
ological response such as activation of defense mechanisms. Charles et al. (2009)
found changes in the protein content and profile of tomato fruit treated with UV-C
(3.7 kJ/m2) at the mature green stage. In particular, the results showed the synthesis
repression of two proteins detected in senescing control fruit and the induction of
stress- and/or pathogenesis-related proteins—both aspects could be involved in the
reduction of B. cinerea incidence. Moreover, UV-C can affect quality attributes in
relation to species and doses: treated papaya maintained a higher firmness than the
control (Zhao et  al., 1996) while strawberry and apple firmness decreased as the
irradiation dose increased (Yu et al., 1996; Drake et al., 1999).

5.3  CHEMICAL CONTROL OF POSTHARVEST DISEASES

5.3.1 Generally Regarded as Safe
Chemical compounds with low toxicity and generally recognized as safe (GRAS)
compounds have received increasing interest for the control of postharvest fruit dis-
ease. GRAS is a US Food and Drug Administration designation and denotes a sub-
stance added to food that is considered safe by experts and for this reason is exempt
from the usual Federal Food, Drug, and Cosmetic Act food additive tolerance
requirements (Senti, 1981). GRAS compounds are allowed with very few restrictions
for many industrial and agricultural applications by regulations worldwide and offer
a considerable promise in postharvest technology, showing antimicrobial, antifun-
gal, and insecticidal properties (Gregori et al., 2008; Fagundes et al., 2013). Peracetic
acid, K-sorb, sodium bicarbonate, and calcium salts are some examples of GRAS
widely used in the food industry for leavening, pH control, taste and texture develop-
ment. They demonstrate a broad spectrum of antimicrobial activity and have been
shown to inhibit the development of postharvest diseases such as blue mold apple
decay (Janisiewicz et al., 2008), M. laxa and Rhizopus stolonifer of stonefruits (Mari
et al., 2004) and green mold and sour rot of citrus (Smilanick et al., 2008). In spite of
interesting results obtained in laboratory and small-scale experiments, commercial
usage limitations arise from inconsistent activity and limited persistence, lack of
preventive effect, risk of fruit injury (Larrigaudiere et al., 2002; Palou et al., 2002).
Physical and Chemical Control of Postharvest Diseases 95

However, preharvest salt treatments (a few days before harvest) seem to overcome
these issues, completely inhibiting the incidence of decay in citrus fruits (Youssef
et  al., 2012) and have been shown a higher efficacy or similar efficacy to that of
conventional chemical treatments against storage rots of table grapes (Nigro et al.,
2006). The antifungal activity of salts against several pathogens is correlated to a
direct inhibition of spore germination, germ-tube elongation and production of pecti-
nolytic enzymes (Hervieux et al., 2002). However, indirect effects must also be taken
into consideration, such as a possible increase of tissue resistance with structural
changes in cell-wall or phytoalexin production induced, for example, by sodium car-
bonate (Venditti et al., 2005). In addition, the osmotic stress caused by the presence
of salts in field applications could contribute to a decrease in epiphytic fungal popu-
lations, including Pencillium spp. species (Youssef et al., 2012).
Other chemicals, such as chlorine dioxide, hydrogen peroxide, citric acid, and
ethanol, are listed as GRAS substances, and their application in the postharvest
phase, by fogging, could be very useful, since some fruits like strawberries require
that handling and wetting are minimized. Vardar et al. (2012) obtained a significant
reduction of postharvest decay in strawberries treated by fogging with hydrogen per-
oxide (2000 μL/L), which resulted in reduced microbial populations on the surface
of the treated fruit and in the storage atmosphere. Moreover, fruit and vegetables are
considered major vehicles for transmission of food-borne enteric viruses since they
are easily contaminated at pre- and postharvest stages and the sanitizers commonly
used are relatively ineffective for removing human norovirus surrogates from fresh
produce. Some GRAS compounds, such as polysorbates, were able to achieve a 3-log
reduction in virus titre in strawberries and an approximately 2-log reduction in virus
titre in lettuce, cabbage, and raspberries (Predmore and Li, 2011).
In 1997, ozone was declared to be GRAS for food contact applications (EPRI
Expert Panel, 1997) and since then ozone-based treatment of fresh fruit and vegeta-
bles has been used in the postharvest handling industry with satisfactory results. In
a preliminary report, the inhibition of conidia of B. cinerea, M. fructicola, P. digita-
tum, and R. stolonifer required an ozone concentration of more than 200 μL/L under
humid conditions, and 4000 μL/L under dry conditions (Margosan and Smilanick,
1998). Unfortunately, it was found that the concentrations of ozone that inactivated
conidia were relatively high and could not be used without complete containment of
the gas and protection of workers. Under conditions where ozone is present during an
8 h workday, gas concentrations cannot exceed 0.075 μL/L (USEPA, 2008). Ozone
was extensively tested for the control of table-grape decay. Although it is fungistatic,
dose-dependent, and can be phytotoxic at high concentrations (above 5000 μL/L), a
treatment with 5000 μL/L ozone in a commercial chamber reduced grey mold inci-
dence from natural inoculum by about 50% after six weeks’ storage at 0°C (Gabler
et al., 2010). Similarly, kiwifruit continuously exposed for four months to gaseous
ozone (0.3 μL/L) showed a delayed development of stem-end rot and a 56% reduc-
tion of grey mold incidence. The observed disease suppression strongly suggests
that ozone treatments induce resistance of kiwifruit to the pathogen. Measurements
of antioxidant substances and antioxidant activity on fruit exposed to ozone for the
same time intervals showed a strong negative correlation between disease incidence
or severity and phenol content (Minas et al., 2010).
96 Advances in Postharvest Fruit and Vegetable Technology

5.3.2 Electrolyzed Water
Electrolyzed water (EW) is generated by the electrolysis of salt solution through an
electrolytic cell where the anode and cathode are separated by nonselective mem-
branes. EW was initially developed in Japan as a medical product and was success-
fully applied as a sanitizer to inactivate microorganisms on food and food processing
equipment surfaces (Al-Haq et al., 2002). Several studies have also documented the
strong antifungal activity of EW against postharvest diseases such as brown rot in
peaches (Guentzel et al., 2010), green mold in tangerine (Whangchal et al., 2010) and
blue mold in apple (Okull and Laborde, 2004). In all of these investigations, elec-
trolysis was conducted with the addition of sodium chloride as the electrolyte, with
a consequent formation of free-chlorine and chlorinated organic compounds like
chloramines, dichloramines and trichloromethanes, creating drawbacks for handlers
and consumers. Moreover, free chlorine is quickly inactivated by the heavy inor-
ganic load present in the wash-water of commercial packing houses; therefore, the
use in the electrolysis reaction of salts not containing chlorine might be particularly
interesting (Fallanaj et al., 2013). Compared to other conventional methods of disin-
fection, EW reduces the treatment time, is easy to obtain, has very few side effects,
and is relatively cheap (Tanaka et al., 1999). In addition, it does not negatively affect
the organoleptic properties, color, scent, flavor, or texture of the various food com-
modities (Al-Haq et al., 2005). However, the main advantage of EW is its safety for
humans and the environment; when it comes into contact with organic matter, or is
diluted with tap-water or water produced by reverse osmosis, it reverts to normal tap
water (Huang et al., 2008). In 2002, Japan officially approved EW as a food additive
(Al-Haq et al., 2005).
The main mechanism of action of EW relates to oxidation that could damage
cell membranes, create disruption in cell metabolic processes and, essentially, kill
the cell. Spore treatment with EW involves cell structural changes as well as reac-
tive oxygen species (ROS) accumulation, mitochondrial membrane integrity and
ATP production. A significant increase of ROS accumulation was observed in P.
­digitatum spores exposed to the electrolyzed salt solution for 15 min. However, other
factors can be involved in the production of ROS, such as pH increase; in fact, the
pH gradient was in the range 8.5–9.0 after the electrolysis process (Fallanaj, 2012).

5.4  NATURAL COMPOUNDS

5.4.1  Volatile Organic Compounds from Plants
Plants produce an amazing diversity of secondary metabolites (more than 100,000
have been identified and at least 1700 are volatile) having a wide range of biologi-
cal activities (Boulogne et al., 2012; Bitas et al., 2013). Some secondary metabolites
occurring in plants in their active forms or as inactive precursors, are associated
with the defense system and have shown potential for the control of postharvest
diseases. Many have been shown to directly inhibit pathogens by disrupting or dam-
aging fungal membranes (Fallik et al., 1998; Arroyo et al., 2007), but some, such
as flavonoids, enhanced the defense responses of the host (Sanzani et  al., 2010).
Physical and Chemical Control of Postharvest Diseases 97

Plant volatile organic compounds (VOCs) are substances with low molecular weight
and high vapor pressure that are naturally emitted by different organs (i.e., leaves,
buds, flowers, fruits, bark, wood, roots). Their high volatility at ambient temperature
makes them suitable for postharvest biofumigation, a technique for disease-control
that offers the advantage of minimal handling and absence of fruit-wetting. Studies
carried out in the last 20 years have produced significant progress in our knowledge
of the antifungal activity of plant metabolites, and more than 20 volatile compounds
from edible plants (spices, herbs, fruits, vegetables) were found to be particularly
interesting as novel means for decay-control because of their safety at low concentra-
tions. Most of these compounds are also widely used as food additives, and the Joint
FAO/WHO Expert Committee on Food Additives expressed no safety concerns at
current levels of intake for allyl-isothiocyanate (AITC), p-anisaldehyde, carvacrol,
(–) carvone, trans-cinnamaldehyde, hexanal, trans-2-hexenal, 2-nonanone, terpineol
and thymol, when used as flavoring agents. Different forms of application (liquid
or vapor phase) and measurements of pathogen inhibition (mycelial growth and/or
conidial germination) applied in the studies often make it difficult to compare the
minimal inhibitory concentrations (MICs) obtained. The most consistent fungicidal
activity by plant bioactive compounds was found with some isothiocyanates (ITCs),
followed by trans-2-hexenal, trans-2-nonenal, carvacrol, thymol, citral and trans-
cinnamaldehyde and they have been reviewed by Mari et al. (2011). The main fac-
tors involved in the antimicrobial activity of the compounds proved to be functional
groups, hydrophobicity and vapor pressure (Andersen et al., 1994; Caccioni et al.,
1997; Arfa et al., 2006). The in vitro inhibition by plant compounds has not always
been confirmed in in vivo assays. Besides chemical characteristics, other factors
proved to influence the effectiveness of antifungal compounds in disease-control,
including treatment conditions (form of application, concentration, temperature,
exposure time, time of treatment, formulation) and characteristics of the pathogen
(age and form of infection structures, location of pathogen in host tissue). Different
levels of sensitivity to treatments were found among fruit species or cultivars, and
detrimental effects on sensory traits (odor, texture, and flavor) or phytotoxic symp-
toms on fruit have been observed in some studies with treatments effective in disease
control (Vaughn et al., 1993; Neri et al., 2006c, 2007; Mehra et al., 2013).

5.4.2 Isothiocyanates
Isothiocyanates (ITCs) derive mainly from hydrolysis of Brassica carinata defatted
meal containing glucosinolates. The ITCs have shown strong activity against a wide
range of food pathogens in specific biological tests (Delaquis and Mazza, 1995).
Some ITCs are volatile substances and could potentially be successfully employed in
treatments such as biofumigation. The postharvest phase, characterized by restricted
environment parameters such as temperature, relative, humidity, and composition of
atmospheric gas, represents an advantage for biofumigation of fresh fruit before stor-
age. In in vitro tests, allyl-isothiocyanate (AITC), a volatile ITC, showed significant
inhibition of conidia germination and/or mycelial growth of M. laxa, P. expansum
(Mari et  al., 1993), Fusarium oxysporum (Ramos-Garcia et  al., 2012), B. cinerea
(Ugolini et  al., 2014) while benzyl ITC inhibited Alternaria alternata mycelial
98 Advances in Postharvest Fruit and Vegetable Technology

growth (Troncoso-Rojas et al., 2005). In in vivo trials, ITCs were found to be active
against numerous postharvest pathogens and on different hosts. However, their activ-
ity did not always confirm the results obtained in preliminary in vitro tests, showing
that the treatment conditions should often be established not only in relation to the
active substance and fungal pathogen but also to the fruit and vegetables response
to treatment (Mari et al., 2008). While many ITCs have been synthesized, little data
has reported on the activity of ITCs produced in situ, although their effectiveness
was similar; in fact, synthetic and glucosinolate-derived AITC vapors were evalu-
ated against B. ­cinerea on strawberries and no significant differences were found
between two origins (Ugolini et al., 2014). This is an important aspect, showing that
biofumigation could be used for industrial applications; the use of bio-based chemi-
cals obtained from renewable natural resources also fits well with the goals of a
sustainable agriculture system. The mechanism by which ITCs inhibit fungal growth
is not yet completely known. Probably, a nonspecific and irreversible interaction of
the ITC with the sulphydryl groups, disulphide bonds and amino groups of proteins
and amino acids residues can take place (Banks et al., 1986). Despite much data on
antifungal, antibacterial, anti-nematode and anti-insect activities of ITCs, only a few
investigations reported their effects on treated fruit quality and residue content. The
postharvest quality of bell peppers represented by general appearance (absence of
phytotoxic symptoms), weight loss, firmness, titratable acidity, and total soluble sol-
ids, was not affected by the mixture of ITC treatment (Troncoso-Rojas et al., 2005).
Similar results were obtained in strawberries, where total phenolic content and anti-
oxidant capacity estimated in treated and untreated fruits showed no significant dif-
ference. In addition, residue analysis performed on fruit at the end of storage (7 days
after treatment) showed very low values (<1 mg/kg) (Ugolini et al., 2014).

5.4.3  trans-2-Hexenal

trans-2-Hexenal is C6 an α,β-unsaturated aldehyde naturally occurring in olive oil,

tea and most fruits and vegetables. The compound is also used as a flavoring agent
and its estimated daily intake in Europe and the United States at 791 and 409 μg/
person per day, respectively, is below the threshold of concern (1800 μg/person per
day), showing no safety concerns at current levels of intake. The compound is known
for its antimicrobial properties and is thought to be involved in the defense mecha-
nism of plants. Its production, together with other C6 aldehydes and alcohols named
“green leafy volatiles”, increases rapidly in damaged plant tissues as a result of the
activation of the lipoxygenase hydroperoxide lyase enzymatic pathway in response
to wounding and herbivore or pathogen attack (Matsui, 2006).
trans-2-Hexenal was shown to have strong antimicrobial activity against many
postharvest pathogens (B. cinerea, C. acutatum, Helminthosporium solani, M. laxa,
Pectobacterium atrosepticum, P. expansum) in vitro and in vivo, and its inhibi-
tion has been found particularly marked against the fungal conidial form (Vaughn
et al., 1993; Fallik et al., 1998; Neri et al., 2006a, 2007; Arroyo et al., 2007; Wood
et al., 2013). The high electrophilic properties of the carbonyl group adjacent to the
double-bond, makes trans-2-hexenal particularly reactive with nucleophiles such as
protein sulphydryl and amino groups of the pathogens. In a study where B. cinerea
Physical and Chemical Control of Postharvest Diseases 99

was exposed to a radio-labeled mixture of cis-2-hexenal and trans-2-hexenal, it was

demonstrated that fungal proteins, and particularly proteins of the surface, were
targets of the C6 aldehydes (Myung et al., 2007). C6 aldehydes were preferentially
incorporated into conidia rather than mycelia, a result correlated to the greater sen-
sitivity of spore germination than mycelial growth to trans-2-hexenal. Postharvest
exposure of pome fruits to trans-2-hexenal vapor, even for short times (2–4 h at
20°C), was found to significantly reduce the infections of P. expansum, while 8 h
treatment was needed to significantly reduce patulin content in “Conference” pears
(Neri et al., 2006a,b). Treatment with trans-2-hexenal showed a curative activity up
to 72 h in control of blue mold, and cold-storage temperature, after exposure of the
fruit to trans-2-hexenal, tested in “Conference” pears, did not affect the activity of
the compound (Neri et al., 2006b). The timing of treatment was particularly impor-
tant. Fumigation with trans-2-hexenal applied immediately after inoculation (2 h)
was effective against M. laxa and B. cinerea, but was generally not useful to control
P. expansum. In “Golden Delicious” apples, P. expansum and patulin content were
greatly reduced by trans-2-hexenal treatment (12.5 μL/L) applied 24 h after inocula-
tion without negative effects on quality traits (Neri et al., 2006c). In contrast, con-
centrations effective in decay-control caused phytotoxic effects in apricots, peaches,
nectarines, “Abate Fetel” pears and strawberries, and they affected fruit flavor in
plums, strawberries, “Conference” and “Bartlett” pears and “Royal Gala” apples
(Neri et al., 2006b,c, 2007).
The corresponding saturated aldehyde, hexanal was shown to inhibit the growth
of several postharvest pathogens in vitro but at concentrations higher than trans-
2-hexenal (Caccioni et al., 1995; Neri et al., 2006a, 2007, 2009; Arroyo et al., 2007;
Song et al., 2007). Fumigation with hexanal (900 μL/L for 24 h at 20°C) significantly
reduced the incidence of decayed fruit in raspberry and blueberry and lesion develop-
ment in peaches artificially inoculated with M. fructicola (Song et al., 2007, 2008).
Although 40 μmol/L of hexanal was effective in killing the majority of P. expansum
spores, “Golden Delicious” apples exposed to this concentration of hexanal for 48 h
showed symptoms of phytotoxicity (Fan et al., 2006). Continued exposure to hexanal
(40–70 μL/L for 7 days) effectively suppressed grey mold in tomato although tomato
respiration increased about 50% and fruit reddening was slowed (Utto et al., 2008).

5.4.4 Carvacrol and Thymol

Carvacrol, a monoterpenoid phenol with a warm and pungent odor, is the char-
acter-impact constituent of oregano essential oils (Origanum vulgare, O. onites,
Thymus capitatus) in which it occurs at concentrations of 60%–80%. It exhibits
fungicidal activity against a wide range of postharvest pathogens (B. cinerea, G.
candidum, M. laxa, N. alba, P. expansum, R. stolonifer) and in particular shows
consistent inhibition of mycelial growth (Plotto et  al., 2003; Neri et  al., 2006a,
2007, 2009). Comparable fungicidal activity was exhibited by the carvacrol isomer
thymol (Plotto et al., 2003). This compound has a strongly aromatic, burnt, medici-
nal odor and occurs mainly in thyme. The antimicrobial activity of carvacrol and
thymol has been ascribed to the hydrophobicity of the compounds, the presence of a
phenolic hydroxyl group in these molecules and an adequate system of delocalized
100 Advances in Postharvest Fruit and Vegetable Technology

electrons (double bonds) that allow the OH group to release its proton (Arfa et al.,
2006). The chemical structure of these molecules would allow these compounds to
act as proton exchangers, reducing the gradient across the cytoplasmic membrane.
The resulting collapse of the proton motive force and depletion of the ATP pool
eventually lead to cell death. Postharvest fumigation with carvacrol or thymol was
effective in controlling B. cinerea and M. fructicola on cherries (Tsao and Zhou,
2000) and M. fructicola on apricots and plums (Liu et  al., 2002). However, they
caused phytotoxic symptoms and off-flavors in cherries and a firmer texture and
phytotoxicity in apricots. Phytotoxicity after carvacrol or thymol exposure was also
observed in oranges (Arras and Usai, 2001) and tomatoes (Plotto et al., 2003). The
addition of a mixture of carvacrol, thymol and eugenol inside active packaging was
effective in reducing decay in table grapes (Guillén et al., 2007) but, as confirmed
also in our unpublished trials on cv Italia, fumigation with these compounds caused
off-flavors in grapes. Exposure to carvacrol vapours failed to control blue mold in
pears and only slightly controlled brown rot in peaches and lenticel rot in apples
(Neri et al., 2006a, 2007, 2009).

5.4.5 Citral
Citral, which occurs naturally as the isomers neral and geranial, is an acyclic α,β-
unsaturated aldehyde mainly contained inside oil glands of lemon and lime peel
and  in the essential oils of many plants. The acceptable daily intake established
for citral by the Joint FAO/WHO Expert Committee on Food Additives (1980)
is ≤0.5 mg/kg body weight and the compound is considered a GRAS compound in
the United States. Nevertheless, the antifungal activity of citral against several post-
harvest pathogens has been well documented in in vitro trials (Wuryatmo et al., 2003;
Palhano et al., 2004; Neri et al., 2006a, 2007, 2009; Zhou et al., 2014). Postharvest
fumigation of fruit with citral showed a low degree of efficacy in the control of
blue mold and brown rot (Neri et  al., 2006a, 2007) and failed to control lenticel
rot (Neri, 2009). Treatment with citral caused severe phytotoxicity in tomato fruits,
either when evaluated as a pure compound or as the main constituent of lemongrass
essential oil (Plotto et al., 2003). As for trans-2-hexanal, the fungicidal activity of
citral has been ascribed to the high electrophilic properties of the carbonyl group
adjacent to the double-bond.

5.4.6  trans-Cinnamaldehyde

trans-Cinnamaldehyde, an aromatic aldehyde with the typical odor of cinnamon, is

the main constituent of essential oils of cinnamon bark and cassia bark and leaves.
Its fungicidal activity against postharvest pathogens (Botryodiplodia theobromae,
C. gloeosporioides, C. musae, Glicephalotrichum microchlamydosporum, M. laxa,
N. alba, P. expansum, P. digitatum, R. stolonifer) has been demonstrated in in vitro
studies (Sivakumar et al., 2002; Utama et al., 2002; Neri et al., 2006a, 2007, 2009).
Exposure of rambutan to trans-cinnamaldehyde vapor significantly reduced fungal
infection without any negative effect on fruit, while treatment in an aqueous solu-
tion of trans-cinnamaldehyde caused phytotoxic symptoms (Sivakumar et al., 2002).
Physical and Chemical Control of Postharvest Diseases 101

Vapor treatment with trans-cinnamaldehyde failed to control blue mold in pears,

brown rot in peaches and lenticel rot in apples (Neri et al., 2006a, 2007, 2009).

5.4.7 Essential Oils
Essential oils are extracts, generally obtained by steam distillation or cold-pressing
various organs of aromatic plants, and contain a complex mixture of compounds (up
to 100) with diverse chemical structures, many of which are volatile. Some essential
oils have shown inhibitory activity on postharvest pathogens although at concentra-
tions usually much higher than single-plant bioactive compounds. Most essential oils
are characterized by one to three main components, which impart the characteristic
odor or flavor to the oil and are generally the bioactive ingredients. The achievement
of essential oils with constant composition could be a critical aspect for their practi-
cal use since quantity and quality of components of essential oils can vary depending
on climate, soil composition, harvest period, chemical polymorphism in populations
and method of extraction, and this may influence the antimicrobial properties of
essential oils. Instead, the possible synergism in antimicrobial activity among differ-
ent components, also occurring as minor molecules, could be an advantage in using
essential oils. In addition, the mixture of a variety of functional groups could reduce
the risk of resistance developed by pathogens.
In studies comparing the antifungal activity of several essential oils, those
containing mainly carvacrol (T. capitatus, O. compactum) or thymol (T. glando-
losus, T. vulgaris, O. vulgare, and Syzygium aromaticum) have been shown to
exhibit the highest inhibitory influences among many postharvest pathogens (A.
citri, B. cinerea, Geotrichum candidum, P. digitatum, P. italicum, R. stolonifer)
(Daferera et al., 2000; Arras and Usai, 2001; Bouchra et al., 2003; Plotto et al.,
2003; Barrera-Necha et al., 2008). Essential oils rich in citral also showed a broad
spectrum of antifungal activity (Shahi et  al., 2003; Palhano et  al., 2004; Lazar-
Baker et al., 2011). Different results with the application of essential oils have been
found in in vivo assays. Exposure to vapors of thyme oil caused severe phytotoxic
symptoms on peaches (Arrebola et al., 2010). Among essential oils rich in citral,
lemongrass (Cymbopogon citratus) completely controlled the development of P.
expansum and B. cinerea infections on apples (Shahi et al., 2003) while it showed
a low reduction of B. cinerea, P. expansum and R. stolonifer infections in peaches
(Arrebola et al., 2010). In addition, the application of lemon myrtle (Backhousia
citriodora) essential oil (250 μL/L) reduced the incidence of M. fructicola rot only
on noninoculated nectarines (Lazar-Baker et al., 2011). Among other essential oils,
spray application of laurel oil (3 mg/mL), containing several components (mainly
1,8-cineole, linalool, terpineol acetate and methyl eugenol), showed good anti-
fungal activity against M. laxa in peaches while it exhibited less control of B.
cinerea in kiwifruit and P. digitatum in oranges and lemons (De Corato et  al.,
2010). Spray emulsion of cinnamon (Cinnamomum zeylanicum) essential oil on
bananas showed better control of crown rot than clove (S. aromaticum) essential
oil (0.2%) while it failed to control anthracnose disease (Ranasinghe et al., 2005).
Vice versa, dip treatment with clove essential oil (50 μg/L) showed a higher effi-
cacy in reducing natural infections than cinnamon oil in papayas (Barrera-Necha
102 Advances in Postharvest Fruit and Vegetable Technology

et al., 2008). Two plant oil-based fungicides have been recently labeled: Sporatec
(Brandt Consolidated, Springfield, IL, containing rosemary, clove and thyme oils)
and Sporan (EcoSmart Technologies, Franklin, TN, containing rosemary and win-
tergreen oils) are now commercially available. However, a study on blueberry to
control A. alternata, B. cinerea and C. acutatum diseases showed that only biofu-
migation with Sporatec resulted in significant reduction of C. acutatum disease; in
addition, both biofumigants negatively affected the sensory quality of treated blue-
berry (Mehra et al., 2013). Some promising results in the disease-control of citrus
fruit were found by incorporation of essential oils in wax coatings. Incorporation
of Lippia scaberrima essential oil (2500 μL/L) in Carnauba Tropical coating (du
Plooy et al., 2009) or Cinnamomun zeylanicum (0.5%) in shellac and/or carnauba
(Kouassi et al., 2012) led to a significant reduction of Penicillium disease in orange
fruit, without detrimental effects on the fruit. An advantage of using coatings with
essential oils could be the close contact between the essential oils and the fruit’s
surface over a long period.

5.4.8  Volatile Organic Compounds from Microorganisms

Recently, increasing interest has also been devoted to biofumigation with VOCs pro-
duced by some microorganisms, such as Muscodor albus (Mercier and Jiménez,
2004; Mercier and Smilanick, 2005; Gabler et  al., 2006), Candida intermedia
(Huang et al., 2011), Streptomyces spp. (Wan et al., 2008; Li et al., 2010, 2012) and
P. expansum (Rouissi et al., 2013), showing fungicidal or fungistatic activity against
a variety of postharvest pathogens. M. albus is an endophytic, nonspore producing
fungus originally isolated from a cinnamon tree. Biofumigation with VOCs pro-
duced by M. albus, applied within 24 h from inoculation-controlled brown rot on
peach, grey mold on apple and table grape (Mercier and Jiménez, 2004; Gabler et al.,
2006), blue mold on apple (Mercier and Jiménez, 2004) and green mold on lemon
(Mercier and Smilanick, 2005), while only if the treatment was applied immedi-
ately after inoculation did it provide some control of sour rot on lemon (Mercier
and Smilanick, 2005). Species of Streptomyces are gram-positive, filamentous and
spore-forming bacteria and volatiles from S. globisporus JK-1 (120 or 240 g/L)
significantly reduced the incidence and severity of blue mold in Shatang mandarin
(Li et al., 2010) and grey mold in tomato (Li et al., 2012). Although P. expansum is
a common postharvest pathogen, VOCs emitted by P. expansum strain R82 com-
pletely inhibited the development of B. cinerea, C. acutatum, and M. laxa (Rouissi
et  al., 2013). Many species of Candida yeasts are effective in the control of fruit
disease and VOCs of C. intermedia C410 isolated from healthy strawberry leaf were
effective in reducing grey mold in strawberry (Huang et al., 2011). Some components
of VOCs emitted from these microorganisms are common to different species of
fungi, bacteria, yeasts or plants, whereas others seem to be unique for one species or
isolate. The antifungal activity of these VOCs seemed to be not related to the relative
abundance of the single components of the mixture. Geosmin, for example, is the
primary component associated with the musty-earthy odor produced by many fungi
and actinomycetes and it has been found to be the most abundant component also
of S. globisporus JK-1; however, minor components (dimethyl trisulphide, dimethyl
Physical and Chemical Control of Postharvest Diseases 103

disulphide and acetophenone) led to complete inhibition of blue mold in “Shatang”

mandarin (Li et al., 2010).
An advantage of the use of volatiles produced by microorganisms is that these
VOCs occur as a mixture of compounds belonging to different chemical classes and
these mixtures could have synergistic or additive properties that cannot be achieved
by any single component alone. Phenylethyl alcohol is probably one of most effec-
tive antifungal components, and is common to several microorganisms, including
M. albus, P. expansum R82, C. intermedia and S. globisporus JK-1. However, when
tested as a single component, it completely inhibited B. cinerea, C. acutatum and
M. laxa mycelium growth and conidial germination at concentrations 2000 times
higher than that naturally released from P. expansum R82 in culture (Rouissi et al.,
2013). Moreover, the quantity and quality of VOC production by some micro-
organisms depend on the growth medium, as reported by Huang et  al. (2012) for
Sporidiobolus pararoseus strain YCXT3. The results suggested that S. pararoseus
is incapable of accumulating highly toxic substances in nutrient yeast dextrose agar
and potato dextrose agar media inhibitory to B. cinerea, while, when it is cultured on
yeast extract peptone agar, it produced VOCs highly effective against both the conid-
ial germination and the mycelial growth of the pathogen. Among 39 VOCs emitted
by yeast and identified using gas chromatography-mass, authentic 2-ethyl-hexanol
was found to have a strong antifungal activity against B. cinerea, suggesting that
the strain YCXT3 of S. pararoseus is a promising agent for the control of grey mold
under air-tight conditions. Disease-control with VOCs produced by microorganisms
is still at experimental/levels and focused on antifungal activity, while the evalu-
ation of effects on fruit quality or residues of treatment in fruit have not yet been
carried out. These aspects should be taken into consideration, since the volatiles
emitted from these microorganisms are characterized by strong odors (for example,
the musty-earthy odor conferred by geosmin). The use of VOCs for postharvest dis-
ease control of fruit on a commercial scale have also to be evaluated in relation to
human safety, and more in-depth investigations on their toxicity for humans have
to be performed. In fact, the occurrence of human asthma symptoms, for example,
has been related to the emission of 2-ethyl-hexanol from dampness-related alkaline
degradation of plasticizer floor material (Norbäck et al., 2000).

5.4.9 Chitosan
Chitosan is an edible coating derived from natural sources by deacetylation of chi-
tin and is considered harmless to humans and the environment. It has been studied
for efficacy in inhibiting decay and extending the shelf-life of various fruits (Aider,
2010). A large amount of data is available on the effectiveness of chitosan in pre- and
postharvest treatments on fresh produce and this was recently reviewed by Shiekh
et al. (2013). On temperate fruit such as strawberries, a chitosan coating controlled
Rhizopus rot and also reduced total aerobic count, coliforms, and weight-loss of fruit
during storage (Park et al., 2005). Similar results were obtained for small bunches of
table grape dipped in 0.5% and 1% chitosan solutions. The treatment decreased the
spread of grey mold infection from a berry to close neighbors (nesting) (Romanazzi
et  al., 2002). The control of brown rot caused by Monilinia spp. was achieved in
104 Advances in Postharvest Fruit and Vegetable Technology

peach and cherry using the application of 0.1% and 1%, respectively, of chitosan
(Li and Yu, 2000; Feliziani et al., 2013). The infections caused by R. stolonifer on
tomato fruits were inhibited after the application of chitosan, although the severity of
soft rot symptoms was not related to the molecular weight of chitosan (Hernández-
Lauzardo et al., 2012). In vitro trials showed the ability of chitosan to reduce mycelial
growth of decay-causing fungi, comparable to the reduction obtained with synthetic
fungicides (Feliziani et al., 2013). However, other results showed high levels of anti-
oxidants, antioxidant activity, ascorbic acid, glutathione and high activity of β-1,3-
glucanase in chitosan-treated strawberries, proving a reinforced microbial defense
mechanism of the fruit and an accentuated resistance against fungal invasion (Wang
and Gao, 2013).

5.5  COMPOUNDS INDUCING RESISTANCE

Several chemical compounds are known for their ability to induce disease-resistance
in treated plants, especially in weedy species, such as cotton (Colson-Hanks et al.,
2012), and sunflower (Tosi et al., 1998), but also in woody plants, like grapevines
(Reuveni et al., 2001). Recently, inducers of resistance have been tested directly on
fruit in order to induce resistance against postharvest fruit pathogens.
β-aminobutyric acid (BABA), a nonprotein amino acid, applied to specific wound
sites on the peel surface of grapefruit is able to induce resistance against P. digita-
tum (Porat et al., 2003). Its activity is concentration-dependent, being most effec-
tive at a concentration of 20 mM. BABA acts directly against pathogens; in fact, at
increasing concentrations of 1–100 mM, it showed a marked reduction in percentage
of P. digitatum conidia germination and germ-tube elongation, in agreement with
other data on B. cinerea and P. expansum on in vitro growth (Quaglia et al., 2011).
However, when BABA was used as a postharvest treatment and fruits were dipped in
the inducer solution, it had no effect or reduced the development of P. italicum and
P. digitatum of orange only slightly (Moscoso-Ramirez and Palau, 2013). Probably,
when BABA is used with the aim of inducing resistance, it does not reduce the
percentage of infections or the lesion diameters; however, it can induce the acti-
vation of chitinase gene expression and protein accumulation (Porat et  al., 2003).
Similarly, acibenzolar-S-methyl (ASM) induced a significant increase in the levels
of PR-1a (antifungal), PR-2 (b-1,3-glucanase), PR-5 (Thaumatin-like protein), and
PR-8 (chitinase) gene transcripts in apple treated with increasing ASM concentra-
tions (Quaglia et al., 2011).
Salicylic acid (SA) is an endogenous hormone having a key role in various
species of plant growth. Positive SA effects have been reported for control of P.
­expansum in sweet cherry (Xu and Tian, 2008), grey mold in peach (Zhang et al.,
2008) and fungal decay in persimmon fruit (Khademi et al., 2012). A stimulation of
the antioxidant enzyme activities reported in sweet cherry treated with SA (2 mM)
suggested that the activation of antioxidant defense plays the main role in resistance
against P. expansum (Xu and Tian, 2008). However, the disease reduction obtained
in treated fruit was very low (<15%) and SA cannot be considered satisfactory and
recommended for inclusion in postharvest decay management programs for fruit
packing houses.
Physical and Chemical Control of Postharvest Diseases 105

5.6  INTEGRATED METHODS

All the alternative methods (physical and chemical) cited above did not always
achieve an acceptable level of postharvest disease-reduction when used individually,
and some had a poor effect against future infection, which can occur after treatment,
or vice versa against established infection before treatment (Droby et al., 2009). To
overcome this drawback, integrated strategies have been proposed and widely inves-
tigated. Treatments with GRAS such as ammonium molibdate or sodium bicarbon-
ate improved the efficacy of some microbial antagonists (Qin et  al., 2006; Torres
et al., 2007), although the effectiveness of combined treatments depends upon the
mutual compatibility, duration, and time at which they are applied. It is postulated
that the enhancement of disease-control is directly caused by the inhibitory effects
of the salts on pathogen growth, and indirectly because of the relatively small influ-
ence of the GRAS compounds on the growth of the antagonist (Qin et al., 2006).
Physical methods like heat could enhance the bioefficacy of microbial antagonists
such as B. subtilis (Obagwu and Korsten, 2003), Pseudomonas syringae (Conway
et al., 2005), Cryptococcus laurentii (Zhang et al., 2007), providing, in some cases,
an equivalent control to synthetic fungicides (Hong et al., 2014).
The combination of HWD and ethanol improved the control of M. fructicola in
peaches and nectarines compared to HWD and ethanol alone (Margosan et al., 1997).
Similarly, a combination of peracetic acid and hot water showed a greater reduc-
tion of brown rot in “Mountain Gold” and “Rome Star” peaches inoculated with M.
­fructicola and treated with 40°C-heated peracetic acid (200 mg/L1) than that observed
in fruit treated only with hot water or peracetic acid (Sisquella et al., 2013). Recently,
Dessalegn et al. (2013) demonstrated a high effectiveness in integrating plant defense
inducing chemicals (PDIC), inorganic salts and hot-water treatments for the manage-
ment of postharvest mango anthracnose. Additive and synergistic increases in effec-
tiveness were also observed by integrating heat therapy with various fungicides, thus
leading to significant reductions in the application of active ingredients (Schirra et al.,
2011). Imazalil (IMZ), a synthetic fungicide employed to control P. italicum and P.
digitatum on citrus, applied at 490 mg/L in aqueous solution at 37.8°C, was found
more effective in decay-control than IMZ in a wax mixture at 4200 mg/L sprayed on
fruit at ambient temperature (Smilanick et al., 1997). On the other hand, the use of a
fungicide in combination with hot water is not always preferable, since pyraclostrobin,
a fungicide belonging to the anilinopyrimidine class, showed residues in orange and
lemons that were dependent on treatment temperature. They approximately doubled
for each 5°C increase in solution temperature above 30°C (Smilanick et al., 2006).
A possible synergistic effect between Debaromyces hansenii, an antagonist yeast,
and UV-C irradiation in controlling brown rot incidence on both artificially inoc-
ulated and naturally infected peaches was observed by Stevens et  al. (1997). The
superiority of the combined treatment was probably due to the ability of UV-C to
control deep-seated infections such as latent infections, whereas the yeast controlled
only superficial infections originating in recent wounds. The integrated treatment of
gamma irradiation and a biocontrol agent (Pseudomonas fluorescens) on “Golden
Delicious” apple against blue mold allows the use of irradiation at lower doses
(200 and 400 Gy) with a dual benefit of no detrimental effects on fruit quality but
106 Advances in Postharvest Fruit and Vegetable Technology

significant control of disease (Ahari Mostafavi et al., 2013). Combined heat and UV
treatment reduced postharvest decays, and maintained kumquat and orange qual-
ity. Heat treatment followed by UV-C radiation was the most effective combination,
while, where UV treatment preceded heat application, the elicitation of phytoalexins
was inhibited (Ben-Yehoshua et al., 2005).

5.7 CONCLUSIONS
Microbial decay is a major factor responsible for postharvest losses and compromises
to the quality of fresh produce. In the past, the use of new fungicides has extended
the shelf-life of fresh fruits by reducing losses, but in the last two or three decades,
concerns about public health and the environment has considerably limited their use
after harvest. Future scenarios are tending increasingly more towards integrated crop
management and organic fruit production, with a consequent reduced use of fungi-
cides, for a sustainable agriculture system. This goal requires new technologies to
control postharvest disease. Intense research in the last 30 years has produced numer-
ous studies that show significant progress in the reduction of pesticide use for disease
control, although some critical points have still to be considered. It is unrealistic to
assume that the physical and chemical methods described above have the same fun-
gicidal activity as pesticides, and an integrated approach appears the best method
to obtain acceptable results. However, further research is needed to investigate the
activity of GRAS compounds, natural compounds and VOCs, in large-scale experi-
ments, their mode of action and their degradation in organisms that are still not fully
understood. Physical methods probably have a better chance of prompt application
on a commercial scale since some of these, like heat, do not require any registra-
tion. Nevertheless, also in this case, more investigations have to provide additional
information on appearance, texture, flavors, and storability of treated fruit. Finally,
research should lead to the development of appropriate tools to tailor a complete inte-
grated disease management strategy specific for each situation that takes into account
factors such as species, climate and seasonal conditions, and the market destination.

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is associated with elevated polyamines and proline levels and alleviates chilling injury in
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Zhao M, Moy J, Paull RE. 1996. Effect of gamma-irradiation on ripening papaya pectin.
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Geotrichum citri-aurantii. Food Control 37: 277–283.
 6 Advances in the
Use of 1-MCP
Chris B. Watkins

CONTENTS
6.1 Introduction................................................................................................... 117
6.2 Effects of 1-MCP on Ripening and Senescence of Horticultural Produce...... 118
6.3 Development of Handling Protocols to Maximize Benefits of 1-MCP......... 124
6.3.1 Apple.................................................................................................. 124
6.3.1.1 1-MCP Concentrations, Exposure Time, and
Application Temperature.................................................... 125
6.3.1.2 Nontarget Materials............................................................ 126
6.3.1.3 Timing of 1-MCP Application............................................ 126
6.3.2 European Pear.................................................................................... 127
6.4 Development of Novel Methods for Application of 1-MCP.......................... 128
6.4.1 Preharvest.......................................................................................... 128
6.4.2 Aqueous and Gaseous Postharvest Application................................ 129
6.4.3 Other Application Methods............................................................... 130
6.4.4 Related Compounds........................................................................... 130
References............................................................................................................... 131

6.1 INTRODUCTION
It is almost 20 years since 1-methylcyclopropene (1-MCP) was discovered and pat-
ented by Ed Sisler and Sylvia Blankenship (Sisler and Blankenship, 1996). 1-MCP
is a cyclopropene that is a competitive inhibitor of ethylene perception that acts by
binding irreversibly to ethylene-binding sites, thereby preventing ethylene binding
and the eliciting of subsequent signal transduction and translation. The process of
discovery of the effects of cyclopropenes and their proposed method of action have
been described (Sisler and Serek, 2003; Sisler, 2006). 1-MCP is extremely active
but unstable in the liquid phase, but is stabilized in a process whereby 1-MCP is
complexed with α-cyclodextrin (Daly and Kourelis, 2000). Floralife Inc. obtained
regulatory approval from the United States Environmental Protection Agency (EPA)
in 1999 for use of 1-MCP on floriculture and ornamental products.
The 1-MCP formulation was first marketed under the name EthylBloc®. The
rights to 1-MCP were subsequently obtained by AgroFresh, formerly a subsidiary of
Rohm and Haas, and now part of Dow Chemicals. EPA approval for use of 1-MCP
on edible food products was obtained in 2002. 1-MCP has several characteristics

117
118 Advances in Postharvest Fruit and Vegetable Technology

that have resulted in rapid approval by regulatory authorities around the world: it is
a gaseous molecule that is easily applied, has an excellent safety profile, leaves no
residues in treated produce, and is active at very low concentrations normally in the
parts per billion range.
The commercialization of 1-MCP as the SmartFreshSM Quality System has led to
rapid adoption of 1-MCP based technologies for many horticultural industries. By
2011, regulatory approval for use of 1-MCP had been obtained in over 40 countries.
1-MCP is registered for use on a wide variety of fruits and vegetables, including
apple, avocado, banana, broccoli, cucumber, date, kiwifruit, mango, melon, nectar-
ine, papaya, peach, pear, pepper, persimmon, pineapple, plantain, plum, squash, and
tomato. The specific products that are registered within each country vary greatly,
relating to the importance of the crop in that country.
As is the case for controlled atmosphere (CA) storage, most use of SmartFreshTM
technology is for apples (Mattheis, 2008; Watkins, 2008). The focus on apples is, in
large part, due to the large volumes of fruit that are kept in CA storage for periods
up to 12 months, depending on the cultivar and growing region. Also, the apple has
been a very suitable fruit for 1-MCP because the ideal product in the marketplace
is one that is close to that at harvest—one with a crisp fracturable texture, and an
acid to sugar ratio appropriate to each cultivar. In contrast, many other climacteric
fruits, such as the avocado, banana, pear, and tomato, require a delay, not an inhibi-
tion of ripening, to ensure that the consumer receives high quality products with
the expected characteristics of color, texture, and flavor. Research continues to seek
commercially useful solutions to managing application of 1-MCP on these fruits.
Even for the “apple,” commercial experience has highlighted that each cultivar is
unique, often with specific challenges, and that these can be variable according to
growing region and industry size and marketing plans. Also, 1-MCP has provided a
tool that has greatly increased understanding of the involvement of ethylene in ripen-
ing and senescence processes.
Consequently, a vast international literature has been developed about 1-MCP and
its effects on fruits and vegetables, and several reviews are available—for example,
Blankenship and Dole (2003), Huber (2008), Sisler (2006), Sisler and Serek (2003),
Toivonen (2008), Watkins (2006, 2008, 2010). A comprehensive review on 1-MCP
is beyond the scope of a single chapter and this update is therefore focused on three
areas of research where new progress is being made, either on understanding the
effects of 1-MCP or on development of new handling protocols and technologies:

1. Effects of 1-MCP on ripening and senescence of horticultural produce.

2. Development of handling protocols to maximize the benefits of 1-MCP.
3. Development of novel methods for application of 1-MCP.

6.2 EFFECTS OF 1-MCP ON RIPENING AND

SENESCENCE OF HORTICULTURAL PRODUCE
Publications on the effects of 1-MCP on different fruits and vegetables include
at least 34 climacteric fruit and fruit vegetables, 17 nonclimacteric fruit and fruit
vegetables, and 31 vegetables (Table 6.1). Sometimes, no effects on ripening and
Advances in the Use of 1-MCP 119

TABLE 6.1
A Summary of Climacteric and Nonclimacteric Fruit and Vegetables That
Have Been Treated with 1-MCP with Selected References
Produce Type Reference

Fruit and Fruit Vegetable—Climacteric

Apple [Malus sylvestris (L) Mill. var.domestica Fan et al. (1999), Watkins and Nock (2012), Watkins
(Borkh.) Mansf.] (Malus x domestica L.) et al. (2000)
Apricot (Prunus armeniaca L.) Cao et al. (2009), Dong et al. (2002)
Avocado (Persea Americana Mill.) Adkins et al. (2005), Dauny et al. (2003), Hayama
et al. (2008), Meyer and Terry (2010)
Banana (Musa acuminata, AAA Group) Golding et al. (1998), Ketsa et al. (2013), Pinheiro
et al. (2010)
Blueberry, highbush (Vaccinium Chiabrando and Giacalone (2011), DeLong et al.
corymbosum L.) (2003), MacLean and NeSmith (2011)
Cherimoya (Annona cherimola Mill.) Hofman et al. (2001), Li et al. (2009)
Chinese bayberry (Myrica rubra Siebold and Li et al. (2006)
Zuccarni)
Custard apple (Annona squamosa L.) Benassi et al. (2003), de Lima et al. (2010b)
Feijoa (Feijoa sellowiana Berg.) Amarante et al. (2008), Rupavatharam et al. (2015)
Fig (Ficus carica L.) Sozzi et al. (2005)
Goldenberry (Physalis peruviana L.) Gutierrez et al. (2008)
Guava (Psidium guajava L.) Hong et al. (2013), Porat et al. (2009), Singh and Pal
(2008)
Jujube (Zizyphus jujube M.) Jiang et al. (2004), Zhang et al. (2012b), Zhong and
Xia (2007)
Kiwifruit (Actinidia deliciosa (A. Chev) C.F. Boquete et al. (2004), Mworia et al. (2011), Vieira
Liang et A.R. Ferguson var. deliciosa) et al. (2012)
Lychee (Litchi chinensis Sonn.) De Reuck et al. (2009), Pang et al. (2001), Sivakumar
and Korsten (2010)
Mamey sapote (Pouteria sapote (Jacq.) H.E. Ergun et al. (2005)
Moore & Stearn)
Mei (Prunus mume Sieb.) Ergun et al. (2005)
Mango (Mangifera indica L.) Sivakumar et al. (2012),Wang et al. (2006, 2009)
Melon (Cucumis melo L.) Ma et al. (2012), Wang et al. (2006, 2009)
Mexican plum (Spondias purpurea L.) Garcia et al. (2011)
Mountain papaya (Vasconcellea pubescens) Balbontin et al. (2007), Moya-Leon et al. (2004)
Muskmelon, Cantaloupe (Cucumis melo L. var. de Souza et al. (2008), Jeong et al. (2008, 2007)
reticulatus)
Nectarine (Prunus persica Lindl.) Bregoli et al. (2005), DeEll et al. (2008b), Ziliotto
et al. (2008)
Papaya (Carica papaya L.) Ahmad et al. (2013), Ergun et al. (2011), Hofman
et al. (2001), Manenoi et al. (2007)
Peach (Prunus persica L. Batsch) Jin et al. (2011), Mahajan and Sharma (2012)
Pear, European (Pyrus communis L.) Chiriboga et al. (2013a), DeEll and Ehsani-
Moghaddam (2011), Gamrasni et al. (2010),
Villalobos-Acuna et al. (2011a)
(Continued)
120 Advances in Postharvest Fruit and Vegetable Technology

TABLE 6.1 (Continued)

A Summary of Climacteric and Nonclimacteric Fruit and Vegetables That
Have Been Treated with 1-MCP with Selected References
Produce Type Reference
Pear, Asian, or Japanese (Pyrus pyrifolia Lee et al. (2012b), Li and Wang (2009), Szczerbanik
Nakai) et al. (2005)
Pear, Laiyang, Yali (Pyrus bretschneideri Rehd.) Fu et al. (2007), Li et al. (2013), Liu et al. (2013)
Persimmon (Diospyros khaki L.) Ahn and Choi (2010), Choi et al. (2013), Harima
et al. (2003), Oz (2011)
Plum (Prunus salicina L.; Prunus x Candan et al. (2006, 2011), Sharma et al. (2012),
domestica L.) Steffens et al. (2013)
Plumcot (Prunus salicina Lindl. x Prunus Lim et al. (2013)
armeniaca L.)
Sapota (Manilkara achras Mill.) Bhutia et al. (2011)
Soursop (Annona muricata L.) de Lima et al. (2010a), Espinosa et al. (2013)
Tomato (Solanum lycopersicum L.) Baldwin et al. (2011), Sabir and Agar (2011), Su and
Gubler (2012); Zhang et al. (2009b)

Fruit and Fruit Vegetable—Nonclimacteric

Cherry (Prunus avium L.) Gong et al. (2002), Mozetic et al. (2006)
Chayote (Sechium edule (Jacq.) Sw.) Cadena-Iniguez et al. (2006)
Cucumber (Cucumis sativus L.) Lima et al. (2005), Nilsson (2005)
Grape (Vitis vinifera L.) Chervin and Deluc (2010), Chervin et al. (2008),
Tesniere et al. (2004)
Grapefuit (Citrus paradisi Macf.) Dou et al. (2005), McCollum and Maul (2007)
Lime (Citrus latifolia Tanaka) Kluge et al. (2003)
Loquat (Eriobotrya japonica Lindl) Cao et al. (2010), Edagi et al. (2011), Cai et al. (2006)
Mandarin (Citrus reticulata L.) Li et al. (2012), Salvador et al. (2006)
Orange (Citrus sinensis L. Osbeck) Porat et al. (1999)
Pepper, hot (Capsicum frutescens L.) Huang et al. (2003)
Pineapple (Ananas comosus L.) Dantas et al. (2009), Selvarajah et al. (2001)
Pitaya (Selenicerus megalanthus Haw) Serna et al. (2012)
Rose apple (Syzygium jambos Alston) Plainsirichai et al. (2010)
Strawberry (Fragaria x ananassa Duch.) Villarreal et al. (2010), Bower et al. (2003), Jiang
et al. (2001), Ku et al. (1999)
Sweet red and green bell pepper (Capsicum Cao et al. (2012), Fernandez-Trujillo et al. (2009),
annuum L.) Ilic et al. (2012)
Watermelon (Citrullus lanatus Thunb.) Mao et al. (2004)
West Indian lime (Citrus aurantifolia Swingle) Win et al. (2006)

Vegetable—Leafy, Root, and Herbs

Asparagus (Asparagus officinalis L.) Liu and Jiang (2006), Zhang et al. (2012a)
Bamboo shoot (Phyllostachys praecox f. Song et al. (2011)
prevernalis)
Basil (Ocimum basilicum L.) Aharoni et al. (2010), Hassan and Mahfouz (2010),
Kenigsbuch et al. (2009)
(Continued)
Advances in the Use of 1-MCP 121

TABLE 6.1 (Continued)

A Summary of Climacteric and Nonclimacteric Fruit and Vegetables That
Have Been Treated with 1-MCP with Selected References
Produce Type Reference
Bean (Phaseolus vulgaris L.) Cho et al. (2008)
Broccoli (Brassica oleracea L.) Fernandez-Leon et al. (2013), Ku et al. (2013), Ku
and Wills (1999)
Cauliflower (Brassica oleracea, L. var. Ma et al. (2011)
botrytis)
Carrot (Daucus carota L.) Fan and Mattheis (2000), Forney et al. (2007),
Kramer et al. (2012)
Chinese cabbage (Brassica campestris L. spp. Porter et al. (2005)
Pekinensis (Lour) Olsson)
Chinese chive scapes (Allium tuberosum Wu et al. (2009)
Rottler ex Sprengel)
Chinese flowering cabbage, Choysum Able et al. (2003)
(Brassica rapa var. parachinensis)
Chinese kale (Brassica alboglabra Bailey) Sun et al. (2012)
Chinese mustard (Brassica juncea var. foliosa) Able et al. (2003)
Chrysanthemum, Garland (Chrysanthemum Able et al. (2003)
coronarium)
Coriander (Coriandrum sativum L.) Hassan and Mahfouz (2012), Jiang et al. (2002)
Eggplant (Solanum melongena L.) Massolo et al. (2011)
Endive (Cichorium intybus L.) Salman et al. (2009)
Ginseng (Panax ginseng C. A. Mey) Park et al. (2013)
Lettuce (Lactuca sativa L.) Fan and Mattheis (2000), Saltveit (2004), Tay and
Perera (2004)
Mibuna (Brassica rapa var. nipposinica) Able et al. (2003)
Mint (Mentha longifolia L.) Kenigsbuch et al. (2007)
Mizuna (Brassica rapa var. nipposinica) Able et al. (2003)
Okra (Hibiscus esculentus L.) Huang et al. (2012)
Onion (Allium cepa L.) Chope et al. (2007), Cools et al. (2011), Downes
et al. (2010)
Pak choy (Brassica rapa var. italica L.) Able et al. (2002, 2003, 2005)
Parsley (Petroselinum crispum Mill.) Lomaniec et al. (2003), Ouzounidou et al. (2013)
Potato (Solanum tuberosum L.) Cheema et al. (2013), Lulai and Suttle (2004), Prange
et al. (2005)
Rocket leaves (Eruca sativa (syn. E. vesicaria Koukounaras et al. (2006)
subsp. sativa (Miller) Thell., Brassica eruca L.))
Spinach (Spinacia oleracea L.) Grozeff et al. (2010)
Summer squash (Cucurbita maxima var. Massolo et al. (2013)
Zapallito (Carr.) Millan)
Tatsoi (Brassica rapa var. rosularis (L.) Makin.) Able et al. (2003)
Tsai Tai (Brassica chinensis) Wang et al. (2014), Zhang et al. (2010a)
Water bamboo shoot (Zizania caduciflora L.) Song et al. (2011)
122 Advances in Postharvest Fruit and Vegetable Technology

senescence have been detected, but an absence of effects is not common even for
nonclimacteric fruits and vegetables.
Several generalizations can be made about responses of fruits and vegetables to
1-MCP, based on the aforementioned reviews, especially those of Huber (2008) and
Watkins (2006, 2010), together with more recent research:

1. The primary features of ripening in climacteric fruits such as softening,

color development, and volatile production, are inexorably linked to eth-
ylene production, but the specific effects of 1-MCP treatment are closely
linked to species, cultivar, and maturity. The capacity to interrupt the pro-
gression of ripening, once initiated, has been found to vary by fruit and the
attributes that have been studied.
2. Nonclimacteric fruit can be affected by 1-MCP, and are providing insights
about the occurrence of ethylene-dependent and ethylene-independent
events during ripening, including changes of gene expression (up- and
down-regulation). Common benefits of 1-MCP treatment on nonclimacteric
products include delayed chlorophyll and protein losses.
3. While the effects of 1-MCP on specific physiological changes can be highly
variable, responses to 1-MCP are typically dependent on “concentra-
tion × exposure time.” If 1-MCP concentrations and exposure periods are
appropriate for the product, the final quality attained in the ripened fruit is
similar to the untreated produce.
4. Losses of health-promoting compounds such as vitamin C are usually
slower in 1-MCP treated produce, whereas effects on phenolic compounds
are often less. Levels of health-promoting compounds in treated fruit are
usually close to those of untreated fruit if they attain full ripening after
1-MCP-induced delays in ripening.
5. Physiological disorders that are associated with senescence or induced by
ethylene (endogenous and exogenous) are inhibited, while those associated
with carbon dioxide (CO2) in the storage environment are often increased.
Chilling injury is increased or decreased depending on whether inhibition
of ethylene production is related to enhancing or alleviating the specific
disorder.
6. Pathological disorders can be increased, decreased, or unaffected by 1-MCP
treatment, the effects likely associated with the interaction of the specific
pathogen with the product (e.g., species and its maturity or ripeness stage)
and the environment.

A major effort has gone into understanding the basis of variation in responses
of species and cultivars to 1-MCP. Peaches show relatively limited inhibition in
ripening after treatment (Dal Cin et al., 2006; Hayama et al., 2008; Ortiz et al.,
2011), while tomatoes respond to treatment even after ripening has started, albeit
to a reduced extent (Hoeberichts et  al., 2002; Hurr et  al., 2005; Tassoni et  al.,
2006; Wills and Ku, 2002; Zhang et al., 2009a, 2010b). Avocados and bananas are
recalcitrant to inhibition by treatment, once ripening has been initiated (Adkins
et al., 2005; Golding et al., 1999; Zhang et al., 2011b). In contrast, others such as
Advances in the Use of 1-MCP 123

European pears may not recover from the inhibition in ripening even when treated
with low 1-MCP concentrations (Bai et al., 2006; Chiriboga et al., 2013a; Ekman
et al., 2004).
Differences in 1-MCP responses for cultivars within species have been described
for many fruit, including apples (Bai et al., 2005; Watkins et al., 2000), avocados
(Pereira et al., 2013 ), guava (Porat et al., 2009), pears (Bai et al., 2006), and tomato
(Guillén et al., 2006). In general, early-season fruit with faster rates of metabolism
are less responsive to 1-MCP, but the effect of harvest maturity is also critical. If
fruits are treated with 1-MCP when immature, ripening can be completely inhibited
or abnormal, for example, guava (Bassetto et al., 2005), tomato (Hurr et al., 2005),
and banana (Bagnato et  al., 2003; Pelayo et  al., 2003). For apples, the effects of
1-MCP on quality factors such as firmness generally decline faster in later-harvested
than in earlier-harvested fruit (Bulens et al., 2012; Lu et al., 2013; Mir et al., 2001;
Toivonen and Lu, 2005) or where internal ethylene concentrations (IECs) are high
in fruit at the time of treatment (Jung and Watkins, 2014; Nock and Watkins, 2013;
Watkins and Nock, 2012). Inhibition in the ripening of pear by 1-MCP may be less
pronounced in more mature pears (Chiriboga et al., 2013b).
The reasons for these differences may include ethylene receptors and their
metabolism, 1-MCP diffusivity and reversible and/or irreversible nontarget binding,
1-MCP metabolism, as well as off-gassing. Although information about ethylene
receptors has increased and is reviewed earlier, little is known about the receptors
in relation to 1-MCP responses across species. Ethylene production of the fruit has
been used as an indirect measure of receptivity of ethylene receptors to 1-MCP.
Zhang et al. (2009a, 2010b, 2011b) have hypothesized that the IEC modulates the
sensitivity of climacteric fruit to 1-MCP, although increased efficacy of 1-MCP
applied under hypoxic hypobaria appears more a function of greater 1-MCP ingress
into the fruit than lower IEC (Dong et al., 2013a). Overall, such a model is consis-
tent with continued sensitivity to 1-MCP during ripening of fruit such as tomato,
which have comparatively low IECs, and reduced sensitivity of fruit such as avo-
cado, which have high IECs and markedly reduced sensitivity to 1-MCP.
Uptake of any gas should be affected by factors such as tissue morphology and
cuticular resistance, but notwithstanding issues related to receptor function, most
species respond to 1-MCP if given at an appropriate concentration and time. 1-MCP
concentrations as low as 0.1 µL ⋅ L−1 slowed the ripening of mangoes when applied
under hypobaria, although normal atmospheric treatment was applied for compari-
son (Wang et al., 2006). However, comparison of hypobaria and normal atmospheric
pressure application demonstrated greater efficacy of infiltration into apple, Chinese
pear, and tomato (Chen et al., 2010; Dong et al., 2013a; Kashimura et al., 2010) and,
to a lesser extent, in Japanese pear (Kashimura et al., 2010). However, the response
of peach to 1-MCP was weak even when 1-MCP was applied under low pressure,
indicating that diffusion of 1-MCP through flesh was not a limiting factor (Hayama
et al., 2005). Also, while differences between influx and efflux of 1-MCP for avocado
and apple have been demonstrated (Dauny et al., 2003), there is little evidence that
diffusivity of 1-MCP into horticultural products is restricted.
However, the depletion of 1-MCP in static systems containing fruits and veg-
etables suggests that 1-MCP is interacting with nontarget binding sites or is being
124 Advances in Postharvest Fruit and Vegetable Technology

metabolized. The reversible sorption shown by off-gassing of 1-MCP from avocado

fruit, which is probably associated with high oil content (Dauny et  al., 2003), is
not free diffusion based on comparisons with ethane efflux (Dong et  al., 2013b).
Nanthachai et  al. (2007) investigated 1-MCP absorption in a range of produce
(apple, asparagus, ginger, green bean, key lime, leaf lettuce, mango, melon, parsnip,
plantain, potato, and tangerine) and found that the initial rate of sorption for each
correlated with fresh weight, dry matter, insoluble dry matter, and water weight, but
not soluble dry matter. The correlation between absorption rate and insoluble dry
matter suggests a strong affinity for 1-MCP by cellulosic materials. High rates and
capacities for 1-MCP sorption by asparagus spears and plantain peel are associ-
ated with high lignin concentrations (Choi and Huber, 2009). It seems unlikely that
nontarget binding would affect efficacy of 1-MCP because there is a huge excess
of 1-MCP molecules compared with calculated binding sites –0.5 to 4.3 × 106 mol-
ecules of 1-MCP available per physiological binding site (Nanthachai et al., 2007).
That said, it is possible that diffusion within some tissues within a fruit, for example,
locular tissues of tomatoes, can be limiting (Dong et al., 2013b), and at low 1-MCP
concentrations required to modulate 1-MCP responses may be more important. The
effects of 1-MCP binding to nontarget materials such as wood are discussed in the
next section.
The role of 1-MCP metabolism has not received adequate attention and more
research is warranted. 1-MCP sorption by fresh cut tissue is due to oxidative
metabolism in response to wound-induced production of reactive species (Lee
et  al., 2012a) and maybe less important for whole fruit. However, evidence that
1-MCP cell free homogenates metabolize 1-MCP exists, and 1-MCP sorption to
asparagus spears was reduced by more than 60% under anoxia 0.06 kPa oxygen
(Huber et al., 2010).

6.3 DEVELOPMENT OF HANDLING PROTOCOLS

TO MAXIMIZE BENEFITS OF 1-MCP
As a new technology available for horticultural industries, SmartFreshTM has led to
the development of handling protocols that maximize the beneficial responses of
products to 1-MCP or minimize undesirable ones. Most research has been on apple,
which is used as a primary example here, but European pear is used as an example of
a fruit that requires postharvest ripening to occur in order to meet consumer expecta-
tions for color, flavor, and texture.

6.3.1  Apple
SmartFreshTM is used widely on apple fruit due to its positive effects on a commodity
that is grown widely around the world, stored in large volumes in air and CA storage,
and then shipped and distributed through marketing chains that can be extensive.
Positive responses of apple fruit 1-MCP include reduced ethylene production and res-
piration rates, maintenance of firmness and acidity, and reduced incidence of many
physiological disorders and greasiness (Watkins, 2006). An important aspect of the
Advances in the Use of 1-MCP 125

apple is that the qualities desired by the consumer, such as a crisp fractuable texture
and sugar/acid balance, are similar to those at harvest; therefore, in contrast to many
other fruit types, postharvest ripening is not usually necessary. The effectiveness of
SmartFreshTM in maintaining quality of fruit in the marketplace has been especially
important as texture of fruit can deteriorate rapidly after removal from storage and
during marketing. Integration of SmartFreshTM into the apple industry has affected
use of postharvest chemical treatments, storage temperature regimes, storage atmo-
spheres, storage duration based on fruit maturity at harvest, and poststorage handling
and packing procedures (Mattheis, 2008). Decisions about use of SmartFreshTM
are influenced by growing region, size of the industry, and cultivar characteristics
(Watkins, 2008), including use at the farm-stand level (McArtney et  al., 2011) or
without refrigeration (Jung and Lee, 2009).
The efficacy of ripening inhibition by 1-MCP is affected by cultivar (DeEll et al.,
2008a; DeLong et  al., 2004; Fan et  al., 1999; Jung and Watkins, 2014; Nock and
Watkins, 2013; Rupasinghe et al., 2000; Watkins et al., 2000) and storage method–
air versus controlled atmosphere storage (Bai et al., 2005; Watkins and Nock, 2005;
Watkins et al., 2000). Research has been carried out to address factors that affect
success of 1-MCP treatment, such as its concentration, effects of nontarget materials,
and timing of 1-MCP application and how these factors interact with cultivar, harvest
maturity, and storage treatments.

6.3.1.1 1-MCP Concentrations, Exposure Time,

and Application Temperature
Most research has focused on the effects of 1-MCP concentrations and exposure
times in air. 1-MCP is more effective at higher concentrations but affected by cultivar
and storage type; for example, fruit of “Queen Cox” were firmer when treated with
10 µL ⋅ L −1 than with 0.5 or 1 µL ⋅ L−1 1-MCP but there was little effect of concentra-
tion for “Bramley” (Dauny and Joyce, 2002). Inhibition of ripening of “McIntosh”
was greater in fruit treated with 1 µL ⋅ L−1 than 0.635 µL ⋅ L−1 (DeEll et al., 2008a).
Also, dose responses were found for “McIntosh” but not for three other cultivars, and
only in air and not CA storage (Watkins et al., 2000). The full label rate for 1-MCP is
1 µL ⋅ L −1 in the United States, Canada, and the European Union (EU).
It has been suggested that 1-MCP is better able to reach and attach to the ethyl-
ene receptor sites at the higher temperature (Sisler and Serek, 1997), and for apples,
lower treatment temperatures can require longer treatment times to get maximum
benefit. Temperature is important when application times are shorter, that is, less
than 9–12 h, and depends on the cultivar (DeEll et al., 2002; Kostansek and Pereira,
2003). No effect of treatment of warm fruit on the day of harvest compared with
overnight cooling was found for four cultivars when treated for 24 h (Watkins and
Nock, 2005). It is recognized that 1-MCP applications can be at any temperature,
and that a 24 h treatment time will take into account variations in factors such as
temperature and room leakiness (Kostansek and Pereira, 2003). While infiltration of
1-MCP into apple fruit greatly reduces the exposure time required to inhibit ripen-
ing (Kashimura et al., 2010), such applications are unlikely to have any commercial
significance.
126 Advances in Postharvest Fruit and Vegetable Technology

6.3.1.2  Nontarget Materials

1-MCP is easily absorbed by nontarget materials that are present in commercial
settings. These include wooden and cardboard bins and bin-liner material, and
absorption can be further increased if these materials are wet; in contrast, absorp-
tion by plastic bins or wall-surface materials is low (Ambaw et  al., 2011, 2013a;
Rodriguez-Perez et al., 2009; Vallejo and Beaudry, 2006). These findings suggest
that the efficacy and uniformity of 1-MCP treatment could be compromised by the
presence of nontarget materials. Possible examples include cases where cultivars
such as “McIntosh,” which recover more easily from 1-MCP inhibition of ripening,
and should be treated in plastic bins to maximize the available 1-MCP. Properly
designed stack arrangement, sufficient ventilation and appropriately placed air-cir-
culating units are important for large treatment facilities (Ambaw et al., 2013b).

6.3.1.3  Timing of 1-MCP Application

The effect of delays between harvest and 1-MCP treatment can also vary greatly
among cultivars, reflecting differences of metabolic rates and associated ethylene
production. For example, delays of more than a week between harvest and 1-MCP
treatment did not compromise the retention of firmness and the control of super-
ficial scald, brown core, and decay in late-maturing cultivars such as “Delicious”
and “Fuji” (Amarante et al., 2010; Lu et al., 2009; Tatsuki et al., 2007). However,
1-MCP treatment delays have a greater influence on rapidly ripening cultivars such
as “Anna,” “Cortland,” “McIntosh,” and “Orin” (DeEll et al., 2008a; Lu et al., 2013;
Tatsuki et  al., 2007). Effects of delayed 1-MCP treatment on quality were more
pronounced in air than in CA storage (Watkins and Nock, 2005). The suppliers of
SmartFresh™ provide recommendations of maximum delays of seven days between
harvest and 1-MCP treatment for most apple cultivars (AgroFresh, 2014). However,
the recommended timing is much shorter for “McIntosh,” with the Canadian registra-
tion requirements and the U.S. recommendations being application of 1-MCP within
three days of harvest (AgroFresh, 2014; DeEll et al., 2008a). Rapid CA treatment of
fruit with 1-MCP can reduce the need to apply CA for up to two weeks (Watkins and
Nock, 2012) or even as long as two months (DeEll and Ehsani-Moghaddam, 2012).
The importance of rapid treatment of 1-MCP can cause logistical issues for storage
operators. Strategies that have been developed by the industry have included the use
of purpose-built rooms, tents, and shipping containers that can be used to treat fruit
quickly after harvest.
The SmartFresh™ label was modified in the United States in 2009 and Canada in
2011 to allow multiple 1-MCP treatments. Therefore, 1-MCP can be applied to fruit
more than once while storage rooms are being loaded, or during storage. Early stud-
ies showed little effect of multiple 1-MCP treatments (DeLong et al., 2004; Mir et al.,
2001) but recent research indicates that the repeated treatments of fruit with 1-MCP
can increase maintenance of firmness and acidity (DeEll and Ehsani-Moghaddam,
2013; Lu et al., 2013; Nock and Watkins, 2013). However, while control of superfi-
cial scald, internal browning, decay and senescent breakdown was maximized with
early single and multiple 1-MCP treatments within the first week of cold storage, the
incidences of a flesh browning disorder on “Empire” apples and external CO2 injury
Advances in the Use of 1-MCP 127

could be increased (DeEll and Ehsani-Moghaddam, 2013; Lu et al., 2013; Nock and
Watkins, 2013). In general, incidences of both external and internal CO2 injuries
(Argenta et al., 2010; DeEll et al., 2003; Fawbush et al., 2008) and firm-flesh brown-
ing of “Empire” apples (Jung and Watkins, 2011) are increased by 1-MCP treatment.
CO2 injuries can be effectively controlled by the antioxidant diphenylamine (DPA)
used for control of superficial scald, but DPA has been banned in the EU. Alternative
control methods, such as delaying CA storage and employing lower CO2 concentra-
tions in the early stages of storage, are being investigated.

6.3.2 European Pear
European pear fruit are categorized as summer, for example, “Bartlett,” or winter
pears, for example, “D’Anjou,” “Bosc,” and “Conference.” In general, both types
require a period of cold storage or ethylene exposure to ripen and attain the buttery
and juicy texture expected by the consumer (Villalobos-Acuna and Mitcham, 2008).
1-MCP decreases ethylene production and respiration rates, and delays softening
of the fruit as well as reduces incidences of superficial scald and internal break-
down, but it is challenging to obtain a delay and not complete inhibition of ripening
(Argenta et al., 2003; Bai et al., 2006; Calvo and Sozzi, 2004, 2009; Kubo et al.,
2003; Villalobos-Acuna et  al., 2011a). The best combination of harvest maturity,
1-MCP concentration, application temperature and time, and storage time to ade-
quately control ripening without developing physiological disorders is still uncertain
(Villalobos-Acuna and Mitcham, 2008).
Cultivar and maturity effects are significant factors affecting the responses of
pears to 1-MCP. Bai et al. (2006) found that “Bartlett” pears ripened when treated
with 0.3 µL ⋅ L−1 and kept at temperatures ranging 10–20°C for 10–20 days after stor-
age; the specific regime was related to length of storage time and whether fruit were
air- or CA-stored. In contrast, ripening of “d’Anjou” pears could not be reinitiated
under these conditions and only 1-MCP concentrations as low as 0.05 µL ⋅ L−1 were
satisfactory. In “Bartlett” pears, the same concentration (0.3 µL ⋅ L−1) and treatment
conditions resulted in diverse responses among pears of different maturities and cold
storage periods (Villalobos-Acuna et  al., 2011a). Ripening of “Conference” pears
is blocked by 0.6 µL ⋅ L −1 1-MCP, whereas 0.3 µL ⋅ L−1 1-MCP resulted in soften-
ing in only some pears (Chiriboga et al., 2011). 1-MCP concentrations (0.025 and
0.050 µL ⋅ L −1) that did not prevent ripening of “Conference” pears (Rizzolo et al.,
2005) cannot be applied commercially at such low concentrations. A key to attain-
ing high quality fruit may be the application of 1-MCP at a more advanced maturity
stage; identifying this stage may be difficult among different orchards but is associ-
ated with ethylene production by the fruit (Chiriboga et al., 2013a,b). A similar rela-
tionship to ethylene production has been shown for “Spadona,” a summer pear with
no chilling requirement (Gamrasni et al., 2010).
In commercial settings, another variable is treatment time after harvest.
Environmental factors may also be a factor as responses of the same cultivar can
vary between growing regions (DeEll and Ehsani-Moghaddam, 2011; Villalobos-
Acuna et al., 2011a). When “Bartlett” pears in Ontario, Canada were treated with
0.3 µL ⋅ L −1 1-MCP one day after harvest, softening was compromised compared
128 Advances in Postharvest Fruit and Vegetable Technology

with treatment three and seven days after harvest; since treatments after seven days
provided less control of disorders, three days represented a compromise recommen-
dation (DeEll and Ehsani-Moghaddam, 2012). In contrast, “Bartlett” pears grown in
California in the United States showed little response to a range of treatment times
and temperatures at the same 1-MCP concentration (Villalobos-Acuna et al., 2011a).
Simultaneous application of 1-MCP and ethylene did not have significant effects
on inhibition of “Bartlett” pear ripening caused by 1-MCP treatment (Trinchero
et al., 2004), whereas positive effects were found for “Bartlett” and “Conference”
pears (Chiriboga et al., 2011; Villalobos-Acuna et al., 2011b).
Also, factors such as the cooling method and bin materials that are not so critical
for apples, may be more important with use of lower 1-MCP concentrations required
to delay ripening of pears (Calvo and Sozzi, 2009).

6.4 DEVELOPMENT OF NOVEL METHODS

FOR APPLICATION OF 1-MCP
6.4.1 Preharvest
Premature drop of fruit is a phenomenon that is especially severe in some apple
cultivars such as “Delicious,” “Honeycrisp,” and “McIntosh” and pears such as
“Bartlett,” resulting in loss of fruit before harvest can be completed. Where permit-
ted by registration, drop of apples and pears is often controlled by preharvest sprays
of aminoethyvinylglycine (AVG; ReTain), which inhibits activity of a key enzyme in
ethylene biosynthesis, ACC synthase, (Bangerth, 1978; Schupp and Greene, 2004),
or by naphthaleneacetic acid (NAA), which inhibits abscission but can increase the
ripening rates of the fruit (Marini et al., 1993; Smock and Gross, 1947; Yuan and
Carbaugh, 2007). AVG applications in the field improve the responses of apples and
other fruit to postharvest 1-MCP treatment (Hayama et al., 2008; Moran, 2006). The
availability of 1-MCP has opened up the possibility of an additional technology for
control of such premature drops.
Early attempts at affecting premature fruit-drop by the use of 1-MCP sprays
or as a gas using enclosed plastic bags were not successful (Byers et  al., 2005),
but AgroFresh has developed formulations of 1-MCP for preharvest application
that are registered under the name of HarvistaTM. The technology had been regis-
tered for use in Chile, New Zealand, South Africa, and the United States by 2014.
Preharvest 1-MCP applications may also have the benefit of delaying ripening and
senescence of fruit where undesirable degrees of inhibition occur when posthar-
vest 1-MCP is applied.
Fruit from HarvistaTM treated apple and pear trees show delayed fruit-drop, lower
ethylene production rates and IECs than in untreated fruit, firmer fruit, and lower
starch pattern indices (McArtney et al., 2008, 2009; Varanasi et al., 2013; Villalobos-
Acuna et al., 2010; Watkins et al., 2010; Yuan and Carbaugh, 2007; Yuan and Li,
2008), although effects on maturity indicators are sometimes inconsistent (DeEll
and Ehsani-Moghaddam, 2010; McArtney et  al., 2008, 2009; Villalobos-Acuna
et al., 2010). In addition to effects on slowing the rate of fruit-drop, other benefits of
HarvistaTM include postharvest benefits of decreasing the rates of softening and loss
Advances in the Use of 1-MCP 129

of acidity (DeEll and Ehsani-Moghaddam, 2010; Varanasi et al., 2013; Villalobos-

Acuna et al., 2010; Watkins et al., 2010). Effects of HarvistaTM on fruit responses are
dependent on the concentration (Elfving et al., 2007; McArtney et al., 2008) and tim-
ing of the application before harvest (Elfving et al., 2007; Varanasi et al., 2013; Yuan
and Li, 2008). HarvistaTM also has the benefits of decreasing water-core develop-
ment in apples before harvest and development of superficial scald, soft scald, soggy
breakdown and internal breakdown, of apples and pears after harvest (DeEll and
Ehsani-Moghaddam, 2010; McArtney et  al., 2008, 2009; Villalobos-Acuna et  al.,
2010).
1-MCP has also been applied prior to harvest to other fruit types, usually, but not
always with the HarvistaTM formulation. Ripening and senescence of figs, mango-
steen, plums, and yellow pitahaya fruit were delayed by 1-MCP sprays or fumigation
on the tree (Cock et al., 2013; Freiman et al., 2012; Lerslerwong et al., 2013; Steffens
et al., 2009). Applications of 1-MCP to sweet cherry trees within three days of eth-
ephon treatment had inconsistent effects on reducing ethephon-induced loss in flesh
firmness without inhibiting desirable effects of ethephon on the fruit-removal force
(Elfving et al., 2009).
In general, the effects of HarvistaTM decrease with longer intervals between
treatment and harvest and with increasing time after harvest (Elfving et al., 2007;
McArtney et al., 2009; Varanasi et al., 2013; Villalobos-Acuna et al., 2010; Watkins
et al., 2010). Combined applications of HarvistaTM and NAA or AVG can be more
effective than any of them alone (Yuan and Carbaugh, 2007). Also, combinations
of HarvistaTM and SmartFresh treatments result in more sustained effects on ripen-
ing after harvest than either do alone (McArtney et al., 2008, 2009; Varanasi et al.,
2013; Watkins et al., 2010). Using tomato plants as a model system, MacKinnon et al.
(2009) found that surfactant type influenced 1-MCP effects and that greater spray
volume was more effective; but no effect of the spray nozzle type used was detected.
Few biochemical studies on the preharvest effects of 1-MCP are available. Inhibited
abscission of cotton and citrus leaves by 1-MCP was associated with reduced gene
expression and enzyme activities of endo-β-gluconase and 1-aminocyclopropane-
1-carboxylic acid synthase (ACS) and oxidase (ACO) (John-Karuppiah and Burns,
2010; Mishra et al., 2008; Zhong et al., 2001). 1-MCP treatment resulted in lower
expression of MdPG2 and MdEG1 genes in the abscission zone of “Delicious” apples
compared with AVG treatment, whereas expression of MdACS5A and MdACO1 were
affected similarly by both treatments (Li and Yuan, 2008; Yuan and Li, 2008). Delay
of ethylene production and fruit-softening by 1-MCP was associated with reduced
expression of MdACS1, MdACO1, MdETR2, MdERS1, and MdPG1 (Yuan and Li,
2008). Varanasi et al. (2013) suggested that variations in responses of apple fruit to
HarvistaTM were associated with different regulation patterns of receptor genes (Zhu
et al., 2010).

6.4.2  Aqueous and Gaseous Postharvest Application

Aqueous application of 1-MCP would provide flexibility when sealed rooms are not
available, with selective treatment of fruit to be stored in a room, and a dip or line-
spray while fruit are being packed. Also, it may be easier to apply lower 1-MCP
130 Advances in Postharvest Fruit and Vegetable Technology

concentrations more effectively in the case of fruit such as pear where recovery from
1-MCP is difficult to achieve under the conditions required for gaseous applica-
tion. 1-MCP applied to apples in water resulted in similar physiological responses
(IEC, firmness, and acidity) as the gas form, but required 700-fold higher amounts
of 1-MCP (Argenta et al., 2007). In contrast, the efficacy of an aqueous application
of 1-MCP for 1 min was as high as gaseous application for 9 h for both avocado and
tomato (Choi et al., 2008). Aqueous application of 1-MCP delayed the storage life
of plums even at the ripe stage (Manganaris et al., 2007, 2008) and resulted in lower
anthocyanin concentrations, PAL activity and CI symptoms such as flesh reddening
(Manganaris et al., 2007).
Thus, ripening and associated events were delayed in proportion to the amount
of 1-MCP applied to the different fruit types, indicating that aqueous 1-MCP shows
similar responses as those treated with gaseous 1-MCP. Ripening factors such as
activity of cell-wall associated enzymes, such as polygalacturonase, lycopene, anti-
oxidants, and volatiles of avocado are delayed but recover to reach levels similar to
those of untreated fruit (Choi and Huber, 2008; Pereira et  al., 2013; Zhang et  al.,
2013). The application of aqueous 1-MCP also raises issues of interaction with other
technologies. Sodium hypochlorite solutions and aqueous 1-MCP are fully compat-
ible if 1-MCP is applied first, but chlorine can result in loss of 1-MCP activity in
multiple-use scenario situations (Choi et al., 2009).
Alternative encapsulation systems to α-cyclodextrin complexes include
cucurbit[n]urils (CB[n](where n is the number of glycoluril units), which are bar-
rel-like macrocyclic molecules, which are prepared from glycoluril and formalin
(Zhang et al., 2011a).

6.4.3 Other Application Methods

Polyvinyl acetate sachets provide a method to slowly release 1-MCP into the atmo-
sphere around produce (Lee et al., 2006). 1-MCP can also be incorporated into poly-
mer films and released from the film matrix by high relative humidity (Hotchkiss
et al., 2007). Both concepts provide interesting prospects for application of 1-MCP
to packaged produce but require optimizing of many factors including film char-
acteristics, sachet size, environmental factors, and specific produce items. The use
of 1-MCP in cyclodextrin-based nanosponges tested for floriculture systmes (Seglie
et al., 2011) has not been explored for fruits and vegetable storage but soy protein
1-MCP-releasing pads have been shown to delay tomato ripening and could be useful
for postharvest “in package” treatments (Ortiz et al., 2013).

6.4.4 Related Compounds
Despite the commercialization of HarvistaTM, claims have been made that 1-MCP
application cannot be made outside of a sealed room environment (Goren et al., 2011;
Seglie et al., 2010; Sisler et al., 2009). A range of compounds related to 1-MCP that may
be used to inhibit ethylene responses of plant tissues continue to be identified, includ-
ing 1-alkenes (Sisler, 2008), 1-substituted cyclopropenes (Apelbaum et  al., 2008),
N,N-dipropyl(1-cyclopropenylmethyl)amine, and N,N-di-(1-cyclopropenylmethyl)
Advances in the Use of 1-MCP 131

amine (Seglie et al., 2010; Sisler et al., 2009), and 3-cyclopropyl-1-enlylpropanoic

acid sodium salt (Goren et al., 2011). Whether these compounds will be registered
for use of food products is uncertain.

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 7 Advances in Edible
Coatings
María Serrano, Domingo Martínez-
Romero, Pedro J. Zapata, Fabián Guillén,
Juan M. Valverde, Huertas M. Díaz-Mula,
Salvador Castillo, and Daniel Valero

CONTENTS
7.1 Concept and Types of Edible Coatings.......................................................... 147
7.1.1 Polysaccharides.................................................................................. 148
7.1.1.1 Cellulose............................................................................. 148
7.1.1.2 Pectin.................................................................................. 149
7.1.1.3 Chitosan.............................................................................. 149
7.1.1.4 Starch.................................................................................. 149
7.1.1.5 Alginate............................................................................... 150
7.1.1.6 Aloe Gel.............................................................................. 150
7.1.1.7 Gum Arabic........................................................................ 150
7.1.2 Proteins.............................................................................................. 151
7.1.2.1 Whey Protein...................................................................... 151
7.1.2.2 Gelatine............................................................................... 151
7.1.2.3 Zein..................................................................................... 151
7.1.2.4 Soy Protein.......................................................................... 152
7.1.3 Lipids................................................................................................. 152
7.2 Effects of Edible Coatings on Fruit Properties.............................................. 152
7.2.1 Effect on Fruit Physiology................................................................. 152
7.2.2 Effect on Organoleptic Quality......................................................... 154
7.2.3 Effect on Fruit Bioactive Compounds and Antioxidant Activity......... 158
7.3 Edible Coatings with Natural Antimicrobial Compounds............................ 158
7.4 Concluding Remarks..................................................................................... 161
References............................................................................................................... 162

7.1  CONCEPT AND TYPES OF EDIBLE COATINGS

Edible coating can be defined as a thin layer of edible material formed as a coat-
ing on a food product and is usually applied by immersing the product in a solu-
tion of the coating. This is in contrast to an edible film, which is a preformed, thin
layer of edible material that is placed as a wrapping on the food product (Falguera

147
148 Advances in Postharvest Fruit and Vegetable Technology

et al., 2011). Food industries and packaging manufacturers have joined in efforts to
reduce the amount of food packaging materials, mainly due to environmental and
consumer concerns. The development of edible coatings has received much attention
in recent years due to advantages such as consumption with the food product, some-
times increasing its organoleptic properties and being produced from agricultural
and marine renewable sources, as well as by using several fungal species (ED, 1995,
1998; FDA, 2006; Bourtoom, 2008).
Components of edible coatings can be divided into three categories: hydrocol-
loids, lipids, and composites. Composites generally contain both hydrocolloid com-
ponents and lipids and represent a good strategy for enhancing coating properties by
taking advantage of the properties of both types of components.
The application of an edible coating onto the fruit surface modifies the internal
atmosphere in the same way that do plastic films (Valero and Serrano, 2010), by
increasing the carbon dioxide and lowering the oxygen concentrations. Then, the
effects of edible coatings on internal gas composition and their interactions on qual-
ity parameters must be determined specifically for each fresh produce. The success
of edible coatings for fruits depends mainly on selecting the appropriate coating that
can give a desirable internal gas composition for each specific product (Cisneros-
Zevallos and Krochta, 2002, 2003).
In this chapter, recent trends in edible coatings are summarized with emphasis
on their applications on fresh fruit commodities and their effects on physiological
behavior, organoleptic quality, nutritive aspects, microbial growth, and levels of
­bioactive compounds with antioxidant activity.

7.1.1 Polysaccharides
Polysaccharides are good materials for the formation of edible coating since they
show excellent mechanical and structural properties but they are hydrophilic and
thus have a poor capacity as a barrier against water vapor and gas diffusion. The
principal polysaccharides of interest for edible coatings are cellulose, starch, gums,
pectins, alginate, and chitosan. The linear structure of some of these polysaccha-
rides, for example, cellulose, amylose, and chitosan, renders their films tough, flex-
ible, transparent, and resistant to fats and oils (Dhall, 2013). In the future, other
complex polysaccharides produced by fungi and bacteria such as xanthan, curdlan,
pullan, and hyaluronic acid, will receive more interest.

7.1.1.1 Cellulose
Cellulose is a polymer of d-glucose monomers linked through ß-(1 → 4) glycosidic
bonds. It has low water solubility in nature but water solubility can be increased
by treating cellulose with alkali to swell the structure, followed by reaction with
chloroacetic acid, methyl chloride, or propylene oxide to yield carboxymethyl cel-
lulose (CMC), methyl cellulose (MC), hydroxypropylmethylcellulose (HPMC), or
hydroxypropyl cellulose (HPC). All these cellulose derivatives are water-soluble,
odorless, tasteless, flexible, and transparent, and exhibit higher barrier capabilities
to moisture and oxygen transmission than cellulose itself (Krochta and Mulder-
Johnston, 1997).
Advances in Edible Coatings 149

7.1.1.2 Pectin
Pectins are linear or branched polymers with a high content of galacturonic acid
and may contain as many as 17 different monosaccharides. Four main types of pec-
tins have been structurally characterized: homogalacturonan, rhamnogalacturonan
I, rhamnogalacturonan II, and xylogalacturonan, which differ in both the structure
of the macromolecule backbone and the presence and diversity of side chains. Pectin
is a class of complex water-soluble polysaccharides used to form coatings. It is a
purified carbohydrate product obtained by aqueous extraction of some edible plant
material, usually citrus fruits or apples. Under certain circumstances, pectins form
gels, which have made them a very important additive in jellies, jams, marmalades
and confectionaries, as well as in edible coatings. Pectin is a high-volume and poten-
tially important food ingredient available in high percentages in agricultural waste.
Pectin coatings have been also studied for their ability to retard lipid migration and
moisture loss, and to improve appearance and handling of foods (Moalemiyan et al.,
2011). Due to their hydrophilic nature, pectin edible coatings have low effectiveness
as a water-vapor barrier but good properties as barrier to oxygen and carbon dioxide.
Low methoxyl pectins are often used as edible coatings because of their ability to
form strong gels upon reactions with multivalent metal cations such as calcium. The
incorporation of calcium in polysaccharide edible coatings reduces their water vapor
permeability, making the coatings water-insoluble (Ferrari et al., 2013).

7.1.1.3 Chitosan
Chitin is the second most abundant natural biopolymer after cellulose. It is the major
structural component of the exoskeleton of arthropods and the cell walls of fungi. The
chemical structure of chitin is similar to that of cellulose with 2-acetamido-2-deoxy-ß-
d-glucose monomers attached via ß-(1 → 4) linkages. Chitosan is the deacetylated form
of chitin which is soluble in acidic solutions, in contrast to chitin (Shahidi et al., 1999).
Thus, chitosan is the low acetyl substituted form of chitin and is composed primarily
of glucosamine, 2-amino-2-deoxy-ß-d-glucose, known as (1 → 4)-2-amino-2-deoxy-d-
glucose. Chitosan, is derived from chitin of marine invertebrates and has been used as
an edible coating for its ability to form a good film on the commodity surface and con-
trol microbial growth. Chitosan is now widely produced commercially from crab and
shrimp shell wastes with different deacetylation grades and molecular weights leading
to different functional properties (No et al., 2007). Chitosan is water-insoluble but sol-
uble in weak organic acid solutions. Chitosan derivatives in the form of acetate, ascor-
bate, lactate, and malate are water-soluble. Water-soluble chitosan can also be produced
in the form of oligosaccharide by enzymatic or chemical hydrolysis (No et al., 2007). To
date, chitosan has attracted considerable interest due to its antimicrobial activity (Dutta
et al., 2009) and has widely been used in antimicrobial films. Chitosan has exhibited
antimicrobial activity against a wide variety of pathogenic and spoilage microorgan-
isms, including fungi, and Gram-positive and Gram-negative bacteria.

7.1.1.4 Starch
Starch is in abundance in the plant kingdom and has thermoplastic properties upon
disruption of its molecular structure (Tharanathan, 2003). Starch granules contain two
types of polymeric molecules: amylose, a linear chain of (1 → 4)-α-d-glucopyranosyl
150 Advances in Postharvest Fruit and Vegetable Technology

units, and amylopectin, a larger molecule with a backbone of amylose and highly
branched side units of d-glucopyranosyl linked by α-(1 → 6)-glycosidic bonds.
Starch can form films by the interaction of hydroxyl groups through hydrogen bonds.
Since these interactions are weak, films are brittle with poor mechanical properties,
although a higher proportion of amylose will improve the film characteristics (Campos
et al., 2011). A concentration of amylose over 70% as in amylomaize starches gives
stronger and more flexible films. The branched structure of amylopectin generally
leads to films with poor mechanical properties (decreased tensile strength and elonga-
tion). The substitution of the hydroxyl groups in the molecule weakens the hydrogen
binding ability and thereby improves freeze-thaw stability and solution clarity. An
ether linkage tends to be more stable than an ester linkage (Tharanathan, 2003).

7.1.1.5 Alginate
Alginate is a polymer isolated from brown seaweed (Phaeophyceae) and can form
translucent, glossy, and strong films with low water vapor and oxygen permeability and
high tensile strength. Alginate is a salt of alginic acid and is composed of d-mannuronic
acid and l-guluronic acid, and has the ability to crosslink with divalent ions such as
calcium to form strong films (Sime, 1990). Alginate films are poor moisture-barriers as
they are hydrophilic films, however, the incorporation of calcium reduces water vapor
permeability making alginate films water insoluble. The capacity of hydrocolloid-based
films as water vapor barriers increases as their solubility in water decreases.

7.1.1.6  Aloe Gel

Aloe vera gel has been identified as a novel coating agent to extend the shelf-life of
perishable food crops with good antimicrobial properties, especially as natural anti-
fungal compound (Valverde et al., 2005). Aloe spp. are perennial succulents plants
characterized by stemless large, thick, fleshy leaves that are lance-shaped and have a
sharp apex and a spiny margin. Aloe leaves have yellow latex, which is referred to as
Aloe juice or sap and has a bitter taste. The leaf pulp is the innermost portion of the
leaf and is composed of the parenchyma cells that contain the gel (Steenkamp and
Stewart, 2007). Aloe gel contains polysaccharides, primarily of ß–(1,4)-linked, poly-
dispersed, highly acetylated mannans (acemannan). Many of the medicinal effects
of Aloe leaf extracts have been attributed to the polysaccharides found in the inner
leaf parenchymatous tissue but it is believed that these biological activities should
be assigned to a synergistic action of the compounds contained therein rather than a
single chemical substance (Eshun and He, 2004; Hamman, 2008). Recently, the leaf
characteristics and gel chemical composition of eight Aloe species as well as their
possible use as edible coatings have been described by Zapata et al. (2013).

7.1.1.7  Gum Arabic

Gum Arabic or gum acacia is a dried, gummy exudate from the stems or branches
of the Acacia species. It is the least viscous and most soluble of the hydrocolloids
(Nisperos-Carriedo, 1994) and is used extensively in the industrial sector because of
its emulsification, film-forming, and encapsulation properties. More than half of the
world supply is used in confectionary to retard sugar crystallization and to thicken
candies, jellies, glazes, and chewing gums, although evidences exist as edible coating
Advances in Edible Coatings 151

for fruits. The main gum to be used commercially is derived from Acacia senegal
because of its good emulsification properties (Elmanan et al., 2008).

7.1.2 Proteins
Proteins have received great attention in edible coating research because of their
abundance as agricultural byproducts and food processing residuals. The presence
of reactive amino acid residuals enables proteins to be modified and cross-linked
through physical and chemical treatments to produce novel polymeric structures
(Gennadios, 2002). Protein-based coatings have more interesting mechanical and
barrier properties than polysaccharides. Many protein materials have been tested
including collagen, corn zein, wheat gluten, SPI, fish proteins, ovalbumin, whey pro-
tein isolate and casein (Khwaldia et al., 2004).

7.1.2.1  Whey Protein

Whey proteins from bovine milk have been studied to a great extent because of their
ability to form transparent and flexible coatings that exhibit good barrier and mechani-
cal properties (Krochta, 2002). Whey proteins are globular proteins that remain soluble
after precipitation of casein at pH 4.6 during cheese making. In bovine milk, these
thermolabile proteins consist of mainly α-lactalbumin, β-lactoglobulin, and other pro-
teins present in smaller fractions (e.g., bovine serum albumin, immunoglobulins, and
proteasepeptones). Whey proteins are commercially available as whey protein isolates
or whey protein concentrates, which have protein content of >90 and 20%–85%, respec-
tively (Reinoso et al., 2008). However, whey protein has a hydrophilic nature and lipids
need to be added to the film-forming solution to reduce the water-sensitivity of films.

7.1.2.2 Gelatine
Gelatine is obtained by controlled hydrolysis of the fibrous insoluble protein, colla-
gen, which is the major constituent of animal skin, bones, and connective tissue. The
characteristic features of gelatine are high content of amino-acids, such as glycine,
proline, and hydroxyproline (Dhall, 2013). Gelatine films can be formed from 20%–
30% gelatine, 10%–30% plasticizer (glycerine or sorbitol), and 40%–70% water fol-
lowed by drying the gelatine gel.

7.1.2.3 Zein
Zein includes a group of alcohol-soluble proteins (prolamines) found in corn endo-
sperm and accounts for 50%+ of the total endosperm protein. Zein has been used
intermittently in a number of industrial applications since becoming commercially
available in 1938 but is currently limited to formulations of coating agents for the food
and pharmaceutical industries. Zein has long been recognized for its film-forming
ability and its use as a bioplastic material is of interest because of its environmental
and renewable qualities. Zein is a mixture of several peptides of different molecular
weight, solubility, and charge that are named as zein fractions and classified accord-
ing to their relative mass and solubility as α, γ, ß, and δ-zein. α-Zein, the major frac-
tion (85% of total zein), is soluble in 50%–95% isopropyl alcohol (Wang et al., 2005).
The utilization of corn zein as a structural polymer has been actively investigated
152 Advances in Postharvest Fruit and Vegetable Technology

in the last decades (Park and Chinnan, 1995). Zein films cast from aqueous ethanol
solutions were rated as moderately good with respect to mechanical properties and
moisture and oxygen barrier properties. Zein films plasticized with oleic acid have
exhibited tensile and moisture-barrier properties that make them potentially useful
as biodegradable packaging materials (Rakotonirainy et al., 2001).

7.1.2.4  Soy Protein

Since the 1960s, soy protein products have been used as nutritional and functional food
ingredients in every food category available to the consumer. Recently, soy protein is
being used as an ingredient for elaborating edible coatings. The content of protein from
soybeans (38%–44%) is much higher than the protein content of cereal grain (8%–15%).
Most of the protein in soybeans is insoluble in water but soluble in dilute-neutral salt
solutions (Dhall, 2013). Soy protein consists of two major protein fractions referred to
as the 7S (conglycinin, 35%) and 11S (glycinin, 52%) fraction. Edible coatings based on
soy protein can be produced in either of two ways: surface film-formation on heated soy-
milk or film-formation from solutions of soy protein isolates (SPIs) (Gennadios, 2002).

7.1.3 Lipids
Because of their apolar nature, hydrophobic lipidic substances are used in fruit coat-
ings mainly as a barrier against moisture migration and to improve surface appear-
ance (Lin and Zhao, 2007). Lipid components commonly used in coatings include
natural waxes (e.g., carnauba wax, beeswax, candelilla wax), acylglycerols, and fatty
acids. Additionally, some authors include shellac, which is a natural resin, as an
ingredient of natural coatings for fruits that are not consumed with peel such as cit-
rus fruits, even though it is not included in the GRAS ingredient list, to provide gloss
to food surfaces. Each hydrophobic substance has its own physicochemical proper-
ties, and, thus, edible films based on lipids have variable behavior against moisture
transfer (Morillon et al., 2002). Lipid compounds include neutral lipids of glycerides,
which are esters of glycerol and fatty acids, and the waxes, which are esters of long-
chain monohydric alcohols and fatty acids.
Wax was the first edible coating used on fruits, with the Chinese applying wax
coatings to oranges and lemons in the 12th and 13th centuries. Although the Chinese
did not realize that the full function of edible coatings was to slow down respiratory
gas exchange, they found that wax-coated fruits could be stored longer than non-
waxed fruits (Park, 1999).

7.2  EFFECTS OF EDIBLE COATINGS ON FRUIT PROPERTIES

7.2.1 Effect on Fruit Physiology
The quality of fruit is determined by a wide range of characteristics such as nutri-
tional value, organoleptic quality, processing, and shelf-life. Fruits are classified as
climacteric and nonclimacteric, with climacteric fruits characterized by an increased
rate of respiration and ethylene production early in the ripening process, while in
nonclimacteric fruits those changes do not occur. However, in both types of fruits,
Advances in Edible Coatings 153

parameters related to fruit quality change during postharvest storage and m

­ arketing,
leading to limited storability and shelf-life. The main postharvest changes are in
taste, aroma, skin color, firmness, as well as weight-loss due to transpiration. The
physiological and biochemical activities involved in fruit-ripening and senescence
can be delayed by a range of postharvest treatments (Valero and Serrano, 2010),
including the use of edible coatings. Edible coatings maintain the quality of fruit
and vegetables by forming a film over the produce, which then serves as a partial
barrier to gas transmission and, thereby, creates a modified atmosphere around the
commodity that affects fruit physiology and biochemistry.
The data in Table 7.1 summarizes the findings from a wide range of published
studies on the effect of various edible coatings on ethylene production, respiration

TABLE 7.1
Effects of Edible Coatings on Ethylene Production, Respiration Rate and
Weight Loss in Climacteric Fruit
Climacteric
Fruit Edible Coating C2H4 Respir Wt. Loss Reference
Sapote Wax  ↑   ↑   ↓  Ergun et al. (2005)
Apple–Fuji Shellac  ↓   ↓   ↓  Hagenmaier (2005)
Red delicious Shellac o  ↓   ↓  Hagenmaier (2005)
Gala Alginate ND ND  ↓  Olivas et al. (2007)
Avocado Methylcellulose ND  ↓   ↓  Maftoonazad and
Ramaswamy (2005)
Plum HPMC—Lipid ND ND o Pérez-Gago et al. (2002)
Whey Protein ND ND  ↓  Reinoso et al. (2008)
Aloe spp.  ↓   ↓   ↓  Guillén et al. (2013)
Versasheen™  ↓   ↓   ↓  Eum et al. (2009)
Alginate  ↓   ↓   ↓  Valero et al. (2013)
A. vera + Rosehip oil  ↓   ↓   ↓  Paladines et al. (2014)
Mango Semperfresh™  ↓  o  ↓  Dang et al. (2008)
A. vera  ↓  o o Dang et al. (2008)
Carnauba  ↓   ↓   ↓  Dang et al. (2008)
Tomato Zein, Alginate  ↓   ↓   ↓  Zapata et al. (2008)
Gum Arabic ND ND  ↓  Ali et al. (2010)
Gum Arabic  ↓   ↓   ↓  Ali et al. (2013)
A. vera ND ND  ↓  Athmaselvi et al. (2013)
A. vera, Shellac  ↓   ↓   ↓  Chauhan et al. (2013)
Nectarine A. vera  ↓   ↓   ↓  Ahmed et al. (2009)
A. vera  ↓   ↓   ↓  Navarro et al. (2011)
A. vera + Rosehip oil  ↓   ↓   ↓  Paladines et al. (2014)
Peach Aloe spp.  ↓   ↓   ↓  Guillén et al. (2013)
A. vera + Rosehip oil  ↓   ↓   ↓  Paladines et al. (2014)

Note: Attribute was decreased (↓), increased (↑), unaffected (o), or was not determined (ND) in each study.
154 Advances in Postharvest Fruit and Vegetable Technology

TABLE 7.2
Effects of Edible Coatings on Respiration Rate and Weight Loss in
Nonclimacteric Fruit
Nonclimacteric Wt.
Fruit Edible Coating Respir Loss Reference
Strawberry Wheat gluten ND  ↓  Tanada-Palmu and Grosso (2005)
Starch ND  ↓  García et al. (1998), Mali and
Grossmann (2003)
Chitosan ND  ↓  Han et al. (2004), Gol et al. (2013)
CMC, HPMC ND  ↓  Gol et al. (2013)
Bell pepper Candelilla wax o  ↓  Hagenmaier (2005)
Raspberry Chitosan  ↓   ↓  Han et al. (2004)
Sweet cherry Chitosan acetate  ↓   ↓  Dang et al. (2010)
Alginate  ↓   ↓  Díaz-Mula et al. (2012)
A. vera  ↓   ↓  Martínez-Romero et al. (2006)
A. vera + Rosehip oil  ↓   ↓  Paladines et al. (2014)
Sour cherry Aloe vera  ↓   ↓  Ravanfar et al. (2014)
Mandarin HPMC - Lipid  ↓   ↓  Pérez-Gago et al. (2002)
Pomegranate Starch  ↓   ↓  Oz and Ulukanli (2012)
Table grape A. vera  ↓   ↓  Valverde et al. (2005), Castillo
et al. (2010)
Chitosan  ↓   ↓  Shiri et al. (2013)

Note: Attribute was decreased (↓), unaffected (o), or was not determined (ND) in each study.

rate and weight loss in a wide range of climacteric fruit. It is clear that the different
coatings with polysaccharide, protein, or lipid constituents induced a reduction in
ethylene production and respiration rate, as well as a delay in ripening during stor-
age. In addition, the edible coatings were effective in reducing weight loss, resulting
in net benefit from an economic perspective. Similarly, Table 7.2 shows the effect
of different coatings on reducing respiration rate and weight loss in nonclimacteric
fruits, which also achieved maintenance of quality attributes during storage.

7.2.2 Effect on Organoleptic Quality

In recent years, there is an increasing consciousness of quality, particularly in relation
to the effect of eating fruit on the health of consumers; this greatly demands research
activities with regard to the production of defined quality, the preservation of qual-
ity during marketing, as well as the evaluation of quality parameters and integrating
this into the production processes (Valero and Serrano, 2010). The term “quality” is
related to the degree of excellence and absence of defects of a fresh produce, which
implies either sensory attributes (appearance, color, texture, flavor, and aroma), nutri-
tive (chemical components used to obtain energy), and functional properties (vitamins
and other nonnutrient phytochemicals). Shewfelt (1999) suggested that the inherent
Advances in Edible Coatings 155

produce characteristics determine quality, but consumer acceptability is determined

by their perception and satisfaction. Thus, quality can be oriented to the produce or
to the consumer point of view. It is well documented that during postharvest storage,
there is deterioration in fruit quality, primarily affecting the following traits: color,
firmness, content of total soluble solids (TSS), and total acidity (TA). The application
of edible coatings could modulate changes in these above mentioned parameters and,
in turn, extend the marketability and shelf-life of these perishable commodities.
With respect to firmness, Figure 7.1 shows published data on the effect of dif-
ferent edible coatings on a range of fruit species stored at different temperatures.
In general, fruit with edible coatings showed higher retention of firmness as com-
pared with control fruit, although the effect was dependent on fruit type, storage
temperature, and type of coating. For example, in strawberries that were stored at
11°C for eight days, hydroxypropylmethylcellulose (HPMC) was more effective than
carboxymethyl cellulose (CMC) on retaining firmness (Gol et al., 2013). Similarly, in
tomato, the most effective coating was gum Arabic followed by zein, while smaller
differences were observed when A. vera and starch were used as coatings (Zapata
et al., 2008; Ali et al., 2010, 2013; Chauhan et al., 2013). However, A. vera was very
effective in reducing loss of firmness in nectarine, sweet cherry, and table grape
(Valverde et al., 2005; Martínez-Romero et al., 2006; Ahmed et al., 2009; Navarro

Control
80
Coated

60
Firmness loss (%)

40

20
t c e A en
y A ra

To o al in

ra
a ra
e
ch

e
ec ad a

m ns

To ma ic

ch
at or ns
St erry MC

St ber MC

at

at inat
er

To arab

ze
e

ve
e
rin o M

gu esce
e
t
be star

ar
Pe A. a lgin
.v
u

Pl Plu A. v
.v

To A. a resc
Sw Gr y gl
b yC

w HP

st
m to

To A.
g
ra ry

o
h rbo

o
e
ra err

rr

at
m rb
um m
p

rr

N voc

at
a

m
he

m
b

ta

o
w

A
w
ra

ra

ee
St

ac
St

Fruit species and edible coating

FIGURE 7.1  Firmness loss in fruit with an edible coating and control and after storage.
(Charts were prepared from data given in references cited in Tables 7.1 and 7.2.)
156 Advances in Postharvest Fruit and Vegetable Technology

et al., 2011). It is well-known that cell-wall hydrolytic enzymes cause dramatic loss
of firmness in fruit tissues, the most important being polygalacturonase (PG), cel-
lulase (CL), pectinmethylesterase (PME), and α and β-galactosidases (GAL), among
others (Valero and Serrano, 2010), and, thus, reduction in activity of these enzymes
would lead to reduced postharvest softening. For example, chitosan, as an edible
coating, becomes bound to pectin and thus prevents access of PG to the substrate
and maintains firmness in papaya (González-Aguilar et al., 2009). Cellulase activ-
ity has been also reduced in carambola fruit treated with chitosan, gum Arabic, and
alginate, as well as PME and β-GAL activities (Gol et al., 2013). On the other hand,
edible coatings can be used to modify the internal atmosphere of fruits and, in turn,
delay senescence (Rojas-Grau et al., 2009). Edible coatings create a passive modified
atmosphere that can influence various changes in fresh fruits, such as firmness, and,
in the case of climacteric fruit, inhibit ethylene production (Falguera et al., 2011).
Color change is another important parameter related to organoleptic quality; and
loss of quality can be measured objectively by increases in HunterLab a/b parameter
and decreases in b, Chroma, and Hue angle. Figure 7.2 shows some examples of change
in these color parameters and its relation to coating type and fruit species during post-
harvest storage. From Figure 7.2 it can be inferred that, in general, the application of

Chroma Control
80
Coated
a/b Hue Hue
Changes in color parameter (%)

a/b
60

a
Hue
a/b Chroma Chroma
40
Chroma
Hue b

20
Chroma
y A ra

To o al in

at era
ee ape ten

ra

a
rb ate

m rbo ns

To ma ic

ch
rr rch
C

eA C
ra ry C

gu scen
um m ver

at nat
b
ze
e
ve

To . a esce
M

rin M
M

ar
he A. v

.v
Pe A. a gin

To ara
Sw Gr glu
be sta

st
yC

m to
.

.
w HP

g
re
ta o

A
A or
l
ec ad
y

o
a

o
rr

rr

N voc
St er
rr
be

at

m
Pl Plu

m
b
be

To
w
w

A
tc

at
ra

ra

h
ra
St

St

ac
St

Fruit species and edible coating

FIGURE 7.2  Color changes as Hunter Lab parameters in fruit with an edible coating and
control and after storage. (Charts were prepared from data given in references cited in Tables
7.1 and 7.2.)
Advances in Edible Coatings 157

different edible coatings led to less changes in the color parameter, although efficacy
depended on type of coating, fruit species, and storage conditions. For instance, the
Chroma index increased a 80% in control table grapes, and ≌40% in plum and peach
and only 18% in tomato, while these increases were much lower in fruits treated with
different coatings such as Aloe vera or A. arborescens gel or alginate (Valverde et al.,
2005; Athmaselvi et al., 2013; Guillén et al., 2013; Valero et al., 2013).
The levels of sugars and organic acids are important in determining the taste of
ripe fleshy fruit, and the relative content of these constituents depends on the activ-
ity and the interaction of sugar and acid metabolism (Valero and Serrano, 2010). TA
usually decreases in the fruit flesh during postharvest storage; this is attributed to
organic acids being substrates for the respiratory metabolism in detached produce.
As can be seen in Figure 7.3, all fruit experienced acidity losses during storage, the
magnitude being dependent on fruit species, ranging from 10% in tomato to 70% in
peach. However, in general, the different coatings led to reductions in acidity losses,
the higher effect being found in tomato and plum coated with alginate and also in
plum and peach coated with A. arborescens (Zapata et al., 2008; Guillén et al., 2013;
Valero et al., 2013).
During postharvest, there is also a general increase in the content of TSS, as
has been reported for nectarines, apricots, kiwifruits, and strawberries (Valero and

Control
80 Coated

60
Acidity losses (%)

40

20
tc eA n

ec ad a

h rbor te

To o al in

ch
e
ra
h

m rbo ens
al a

T o m a ic
m ns
ra ry C
C

eA C

at nat
er

r
e

r
er arc

b
ze
e CM

rin M

ve
Pl Plu . ve
t

ar
n
Sw Gr glu

To ara
.v

A A. v

To . a esc
o resc
gi

i
wb HP

st
wb st

m to

To A.
g
o
ra erry

o
ry

at
St rry

m
N oc
p
rr
St er

gu

at

m
a

a
he
wb

m
v
ta

Pe A.
wb

A
at
ra

ra

um
ee
St

ac
St

Fruit species and edible coating

FIGURE 7.3  Acidity loss in fruit with an edible coating and control and after storage.
(Charts were prepared from data given in references cited in Tables 7.1 and 7.2.)
158 Advances in Postharvest Fruit and Vegetable Technology

Serrano, 2010). This increase in soluble solids is much higher in fruits that accu-
mulate larger amounts of starch during development on the plant, such as mango or
bananas. The application of edible coatings on fruit generally leads to lower increases
in TSS, such as in strawberry coated with starch, CMC or HPMC (García et  al.,
1998; Mali and Grossmann, 2003; Gol et al., 2013) and in tomato coated with gum
Arabic or starch (Ali et al., 2010; Das et al., 2013), as a consequence of a delay in
the postharvest ripening process. However, in other reports, higher increases in TSS
have been found in coated fruit than in controls, such as in wax-coated sapote fruits
(Ergun et  al., 2005) and in zein, alginate or A. vera gel-coated tomatoes (Zapata
et al., 2008; Athmaselvi et al., 2013).

7.2.3 Effect on Fruit Bioactive Compounds and Antioxidant Activity

Fruits contain hundreds of nonnutrient constituents with significant biological activ-
ity, generally called “bioactive compounds” or phytochemicals, which have anti-
oxidant activity and protective effects against several chronic diseases associated to
aging, including atherosclerosis, cardiovascular diseases, cancer, cataracts, increased
blood pressure, ulcerous, neurodegenerative diseases, brain and immune dysfunc-
tion, and even against bacterial and viral diseases. These bioactive compounds
vary widely in chemical structure and function in plant tissues and are grouped in
vitamins (C and E), carotenoids, phenolics, and thiols (Asensi-Fabado and Munné-
Bosch, 2010; Baldrick et al., 2011; Brewer, 2011; Serrano et al., 2011; Li et al., 2012;
Valero and Serrano, 2013).
No general tendency has been found for the changes in bioactive compounds
during fruit storage. Thus, loss of compounds beneficial to health, such as pheno-
lics and ascorbic acid, has been found in apples, table grapes, and pomegranates,
while increases in phytochemicals have been observed in sweet cherry and plum
cultivars (Díaz-Mula et al., 2009; Serrano et al., 2009, 2011). Figure 7.4 shows pub-
lished examples in which the content of total phenolics decreased during storage,
and the beneficial effects of the different coatings reduced these phenolic losses.
Interestingly, tomato coated with gum Arabic showed increases in total phenolics
while they decreased in control fruit (Ali et al., 2010, 2013).
Similar behavior was found for total antioxidant activity (Figure 7.4) with lower
losses in chitosan-coated pomegranate, pear and blueberry than in controls (Duan
et al., 2011; Ghasemnezhad et al., 2013; Kou et al., 2014) and even TAA increased
in sweet cherry and tomato coated with alginate and gum Arabic, respectively, more
than in control fruits (Díaz-Mula et al., 2012; Ali et al., 2013).

7.3 EDIBLE COATINGS WITH NATURAL

ANTIMICROBIAL COMPOUNDS
In recent years, new edible films and coatings are being formulated with the addition
of natural antimicrobial compounds for application onto fresh and minimally pro-
cessed fruit commodities. This system constitutes an environment-friendly technol-
ogy and improves the mechanical handling properties that may enhance food quality,
safety, stability, by providing a semi-permeable barrier to water vapor, oxygen, and
Advances in Edible Coatings 159

450
Control Control 400
350
Coated Coated 100
100
80
Changes in phenolics (%)

60

Changes in TAA (%)

50
40

20
0

–50 –20

–40
o

o
te

te

ar
ry
y

y
ar

y
at

at
rr

rr

rr

rr

rr
na

na

pe
er
pe

m
be

he

be

he
ra

wb

ra

wb
to

to
n
n

ue

ue
tc

tc
eg

eg

sa
sa

ra
ra

ic

ic
bl

bl
e

e
m

ito
ito

we

we
st
st

ab

ab
n

n
po

po

Ch
Ch

n
n
sa

sa
ar

ar
es

es
sa
sa
n

n
ito

ito
um

um
at

at
ito
ito
sa

sa
in

in
Ch

Ch
i to
ito

Ch
Ch

G
lg

lg
Ch

Ch
A

A
Fruit species and edible coating Fruit species and edible coating

FIGURE 7.4  Changes in total phenolics or total antioxidant activity (TAA) in fruit with an
edible coating and control, and after storage. (Charts were prepared from data given in refer-
ences cited in Tables 7.1 and 7.2.)

carbon dioxide, between the fruit and the surrounding atmosphere, with increased
antimicrobial properties (Valencia-Chamorro et al., 2011). There is a wide range of
naturally-occurring compounds that exhibit antimicrobial activity, including chito-
san, polypetides, and essential oils or spice extracts. As already stated, chitosan is
a polysaccharide that shows antimicrobial activity, which has been attributed to its
positive charges that would interfere with the negatively charged residues of mac-
romolecules on the cell surface, rendering membrane leakage (Sebti et al., 2007).
Most of the antimicrobials proposed to be used in the formulation of coatings must
inhibit the spoilage microorganisms (bacteria and fungi) and reduce the food-borne
pathogens. In recent years, there is a trend to select the antimicrobials from natu-
ral sources and to use generally GRAS compounds, in order to satisfy consumer
demands for healthy foods, free of chemical additives (Campos et al., 2011).
The essential oils (EOs) have these characteristics. EOs or the so called volatile
or ethereal oils are aromatic oily liquids obtained from plant organs: flower, bud,
seed, leave, twig, bark, herb wood fruit, and root (Serrano et  al., 2008). Natural
compounds that also possess antioxidant effects have been extracted from plants
that belong to genus Thymus, Origanum, Syzygium, Mentha, and Eucalyptus (Burt,
2004). Chemical composition of EOs is complex and strongly dependent on the part
of the plant considered (e.g., seed vs. leaves), the moment of harvest (before, dur-
ing, or after flowering), the harvesting season and the geographical sources. Major
components in EOs are phenolic substances, which are thought to be responsible for
the antimicrobial properties, and many of them are classified as GRAS (Campos
et al., 2011). The antimicrobial activity of the EOs can be attributed to their content
160 Advances in Postharvest Fruit and Vegetable Technology

of monoterpenes that, due to their lipophilic character, act by disrupting the integrity
of microbial cytoplasmic membrane. Lipophilic compounds accumulate in the lipid
bilayer according to its specific partition coefficient, leading to disruption of the
membrane structure (Liolios et al., 2009).
Table 7.3 gives some examples of different coatings on several whole fruits, in
which improvement of coating efficacy was achieved by the incorporation of some
natural antimicrobial compounds, such as EOs from different plant origin. The
most studied parameter was the contamination of different microorganisms such
as bacteria (E.coli, Salmonella spp. and S. aureus) and fungal species (Penicillium,
Rhizopus and Botrytis). Apart from the antimicrobial activity, the combined use
of the edible coatings with natural antimicrobials was also effective in improv-
ing the parameters related to organoleptic and functional quality. As an example,
tomato coated with zein at 10% plus the addition of EOS (thymol, carvacrol, and
eugenol at 75 µL/L) exhibited a reduced rate of color-change than control tomatoes
coated with zein alone after nine days of storage at 10°C (Figure 7.5). The effect
was attributed to the reported antioxidant properties of EOs leading to a delay of
the postharvest ripening process (Serrano et al., 2008). Accordingly, the addition

TABLE 7.3
Improvement of Coating Efficacy with the Addition of Natural Antimicrobial
Compounds
Edible Natural
Fruit Coating Antimicrobial Effects Reference
Blueberry Chitosan 2% Phenolics from Reduced decay and Yang et al. (2014)
blueberry increased phenolics
extracts
Pepper, Pullulan Summer savory Inhibited growth of Kraśniewska et al.
Apple 10% herb Gram-positive and (2014)
Gram-negative bacteria
and P. expansum
Strawberry Alginate 2% Carvacrol, Methyl Inhibited E. coli and B. Peretto et al. (2014)
Cinnamate cinerea
Apple Cassava Cinnamom or Inhibited S. aureus and Oriani et al. (2014)
starch 2% fennel Salmonella
Plum Carnauba Lemongrass oil Inhibited Salmonella and E. Kim et al. (2013)
wax coli, reduced ethylene and
improved quality
Orange Chitosan Tea tree oil Reduced P. italicum growth Cháfer et al. (2012)
Tomato Chitosan Lime essential oil Inhibited Rhizopus Ramos-García et al.
(1%) stolonifer and E. coli (2012)
Table Chitosan or Bergamot oil Reduced microbial counts Sánchez-González
grape HPMC et al. (2011)
Lemon Wax Carvacrol or Reduced P. digitatum, Pérez-Alfonso et al.
thymol respiration and acidity loss (2012), Castillo
et al. (2014)
Advances in Edible Coatings 161

Day 0 Day 0
Control Control
Zein Alginate
100 Zein + EOs Alginate + EOs 8

80

Firmness (N/mm)
Color (Hue angle)

60

4
40

2
20

Tomato Plum

FIGURE 7.5  Color (Hue angle) after nine days’ storage of tomatoes coated with zein at 10%
or zein + essential oils (EOs), and fruit firmness of plums coated with 3% or alginate + EOs
(Serrano and Valero, unpublished data). Data are the mean ± SE.

of these EOs to alginate led to reduced softening, since firmness of plums after
15 days of storage at 2°C was significantly higher in alginate + EOs coated plums
compared with alginate alone or controls, for which an accelerated softening pro-
cess occurred (Figure 7.5).

7.4  CONCLUDING REMARKS

The use of edible coatings for preservation of whole fruit is a matter of high inter-
est taking into account the increasing number of research reports on this issue. For
each particular fruit, the design of an appropriate coating formulation is essential for
assuring the quality and safety during postharvest storage. The proper selection of
an edible coating will depend on the respiration and transpiration rates of the com-
modity and on the environmental conditions of the storage area. Edible coatings can
protect perishable fruits from deterioration during storage by retarding weight loss,
reducing respiration rate and ethylene production, improving texture and other qual-
ity parameters, and reducing microbial contamination. Many of the polysaccharide-
based and protein-based coatings, especially those with inherent antimicrobial or
antifungal activities such as chitosan, are attracting more interest as substitutes for
traditional lipid coatings.
Edible coatings are effective as a barrier to respiratory gas exchange and water
vapor. The efficacy of edible coatings depends on the coating, type and characteris-
tics of the fruit, type of coating, and storage conditions (temperature and duration).
More research is needed in order to get a better understanding of the relationship
between the internal atmosphere produced by the edible coating and the physiological
162 Advances in Postharvest Fruit and Vegetable Technology

changes in fruits during storage that will influence the final quality of the coated
product. The coatings used for one fruit cultivar may not be appropriate for another
since each fruit is different in peel resistance, gas diffusion, and fruit respiration rate
among other attributes. One advantage of using edible coatings is that several active
compounds can be incorporated into the polymer matrix and consumed with the
food, such as the use of natural antimicrobial compounds. In this sense, a new gen-
eration of edible coatings is being currently developed allowing the incorporation of
EOs for controlling spoiling microorganism and thus enhancing the safety of coated
fresh fruits. Finally, sensory evaluation and consumer acceptability tests need to be
conducted during the storage of coated fruit.

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edible coating based on Aloe vera gel to maintain table grape quality and safety. J Agric
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oleic acid films investigated by X-ray diffraction. Mol Biosci 5: 1200–1208.
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 8 Low Ethylene Technology
in Non-Optimal Storage
Temperatures
Ron B.H. Wills

CONTENTS
8.1 Introduction................................................................................................... 167
8.2 Cool Chain Management: The Preeminent Technology............................... 169
8.3 Effect of Ethylene Concentration on Postharvest Behavior.......................... 170
8.4 Interaction of Ethylene and Temperature on Postharvest Behavior.............. 173
8.5 Methods for Inhibiting Ethylene Action........................................................ 174
8.5.1 1-Methylcyclopropene........................................................................ 175
8.5.2 Nitric Oxide....................................................................................... 176
8.5.3 Nitrous Oxide.................................................................................... 177
8.6 Methods for Removing Ethylene from the Atmosphere................................ 178
8.6.1 Ventilation.......................................................................................... 178
8.6.2 Absorption and Oxidation................................................................. 180
8.6.2.1 Potassium Permanganate.................................................... 180
8.6.2.2 Ozone and Active Oxygen.................................................. 181
8.6.2.3 Prestorage Modified Atmospheres...................................... 182
8.7 Environmental Balance................................................................................. 183
References............................................................................................................... 185

8.1 INTRODUCTION
Ethylene (CH2 = CH2) is notable as the defining metabolic difference between plant
and animals systems. Ethylene is only synthesized by plants and has a major regula-
tory role in the growth, development, and senescence of all plants. This is in contrast
to the lack of any known synthesis or metabolic effect on animal systems. In addi-
tion, ethylene was the only known endogenous gas to act as a plant-growth regulator,
but the relatively recent discovery of the synthesis and metabolic effects of the previ-
ously considered toxic gases, nitric oxide (Leshem, 2000), and hydrogen sulphide
(Wagner, 2009) in animals, and even more recently in plants, has underscored the
limitation of our knowledge of metabolism (see Chapter 9 for postharvest effects of
nitric oxide).
Effects of ethylene on fruit and vegetables have been known for many centuries,
even if its identity remained unknown due to inadequate analytical techniques. It is

167
168 Advances in Postharvest Fruit and Vegetable Technology

believed that the ancient Egyptians and Chinese unknowingly stimulated ethylene
production by damaging plant tissues in a confined environment to accelerate the
ripening of figs and pears, respectively. In the intervening years, a wide range of
other effects of ethylene have been recorded, including early in the twentieth century,
when heaters burning kerosene were used to degreen lemons, although the effect was
attributed to the heat rather than the ethylene generated as a byproduct of the incom-
plete combustion of kerosene. It was not until the 1930s that ethylene was identified
as the causative agent with Gane (1934) reporting ethylene synthesis by plants and
Crocker et al. (1935) proposing ethylene as the agent responsible for fruit-ripening
and senescence of vegetative tissues.
The availability of gas chromatography in the 1960s greatly expanded research
efforts into the biosynthesis of ethylene. A major contributor to elucidation of the
metabolic pathway of ethylene was Shang Fa Yang, who had 30 prolific years at UC
Davis on postharvest metabolic studies; the initial stages in the ethylene synthesis
pathway is now commonly referred to as the Yang cycle (Bradford, 2008). The essen-
tial elements in ethylene synthesis start with conversion of the amino acid methio-
nine → S-adenosyl methionine (SAM) → 1-aminocyclopropane-1-carboxylic acid
(ACC) → ethylene. The enzymes catalyzing these reactions are, respectively, methi-
onine adenosyltransferase (MAT), ACC synthase, and ACC-oxidase. The activity of
ACC synthase (ACS) is considered the rate-controlling step for ethylene production.
The final step requires oxygen to release ethylene (Yang, 1985).
It is of considerable importance that while ethylene is synthesized as part of the
natural plant development cycle, it is also induced as a response to a range of external
factors, such as mechanical wounding, pathogen attack, and abiotic environmental
stress. In this respect, production of ethylene is considered to trigger a range of pro-
tective actions by plants, and is thus a beneficial defensive agent (Morgan and Drew,
1997). In addition, ethylene in the ambient air can arise from a range of industrial
sources, such as motor vehicle exhaust or industrial effluent, or from senescing veg-
etation. It can also arise in mixed-load storage or transport where a range of different
produces are held in the same chamber, and ethylene evolved from one produce will
impinge on other lower ethylene emitters in the same chamber. The rapid diffusivity
of ethylene into plant tissues means that exogenous sources of ethylene are as effec-
tive as endogenous ethylene in modifying metabolic and physiological behavior.
The tenet of this chapter is that reducing exposure of fruit and vegetables to ethyl-
ene can be beneficial in extending postharvest life. An obvious treatment is one that
inhibits metabolism of ethylene by produce, but the diffusivity of exogenous ethylene
means that any low ethylene technology must be able to counteract all sources of
ethylene that the produce might encounter during storage and handling.
Any intervention technology needs to recognize the differing role of ethylene in:
(1) initiating ripening in climacteric fruit; and (2) accelerating senescence of noncli-
macteric fruit and vegetables (Abeles et al., 1992; Wills et al., 2007a). Climacteric
produce are fruits with a distinct ripening pattern, with the defining characteristic
being a pronounced increase in respiration to a peak value or climacteric. They are
generally harvested at a mature but unripe state, with the aim of preventing ripening
during storage and transport to markets, by inhibiting initiation of the full ripening
Low Ethylene Technology in Non-Optimal Storage Temperatures 169

process by ethylene. However, such fruit need to be ripened before consumption so

any technological intervention needs to be either reversible or temporary.
Nonclimacteric fruits are harvested at or close to desirable eating quality and do
not exhibit dramatic postharvest changes and show only relatively small changes
in respiration. Horticultural commodities that are not fruits (i.e., nonseed-bearing
vessels) are classed as vegetables, and are nonclimacteric in behavior, as they are
harvested at a commercial maturity stage when desirable eating quality is pres-
ent, although the physiological maturity of vegetables can vary widely (Wills et al.,
2007a). For most nonclimacteric produce, the postharvest regime aims to minimize
any rate of change, and their response to ethylene is an acceleration of the rate of
normal senescence.

8.2 COOL CHAIN MANAGEMENT: THE

PREEMINENT TECHNOLOGY
Over the centuries, the seasonality and perishability of most horticultural crops have
generated interest in methods that would extend the period over which produce are
available in any specific location. The benefit of storing produce in reduced tempera-
tures has long been realized in countries with a cold winter. For example, storage
of Chinese cabbages in China and apples in North America and Europe in cellars
or caves, sometimes ventilated with cool outside air, was practiced at village and
farm level, and provided a much needed food source during winter. The advent of
mechanical refrigeration in the 1850s transformed the use of low-temperature stor-
age from an art into a reliable technology. The first mechanical ice-making machine
was built in 1851 by James Harrison in Geelong, Australia, and was followed by a
commercial refrigeration unit in 1854 that used an ether vapor compression system.
However, interest was in producing frozen meat for export to England rather than the
cool-storage of fruit and vegetables. The first commercial food cold storage facility
in the United States was installed in Boston in 1881, and was followed over the next
10 years by many urban and farm-based facilities (Ryall and Pentzer, 1974; Farrer,
1980). Thus, low-temperature refrigerated storage of food was well established at the
beginning of the twentieth century, with apples and pears being the major horticul-
tural crops to be stored.
In the 1950s, apart from the major commodities, there was still a strong season-
ality of most produce, with consumption generally close to production areas, and
where quality was secondary to availability. In the intervening 60+ years, the horti-
cultural industry has moved to a virtual 12-month availability for many commodi-
ties, which is supported by a substantial international trade of a greater diversity
and quantity of produce. While many factors have had a role in making this change
possible, the major technological impact is considered to be cool-chain management,
where a controlled temperature environment is maintained from farm-storage to
domestic consumption, to inhibit ripening and senescence. Numerous studies iden-
tified the optimum low temperature at which individual produce could be held to
achieve  the maximum postharvest life. The reduction in metabolism achieved by
reducing the temperature should be optimally just above the freezing temperature
170 Advances in Postharvest Fruit and Vegetable Technology

of about −1°C. However, for many produce, the optimum temperature is well above
freezing due to abnormal metabolism, leading to chilling-related injury, physiologi-
cal disorders, or failure to fully ripen when transferred to ambient temperature.
While cool-chain management has become the technology of first resort, its use is
energy-intensive and it utilizes costly infrastructure. Refrigeration became de rigeur
when energy was cheap and greenhouse gas emissions were of no concern. The
world has now moved to an energy minimization mode and horticulture, in com-
mon with many other industries, is aiming to reduce its carbon footprint from an
economic perspective, due to the increasing cost of energy, and to be seen by the
community as a good corporate citizen.
East (2010) reviewed postharvest energy usage of horticultural commodities and
situations, and cites studies that estimated:

1. Cooling and storage of fresh horticultural produce in California uses about

1 billion kWh of energy, which was 5.5% of the electricity used by agricul-
ture in the state (Thompson and Singh, 2008)
2. Domestic and retail refrigeration account for about 2.5% of Britain’s carbon
dioxide emissions (Garnett, 2006)
3. The Australian vegetable industry accounts for 0.7% of national emissions
(O’Halloran et al., 2008)

In addition, refrigeration in America’s supermarkets is the main use of energy,

accounting for 36% of all energy costs (E-source, 2013).
In an energy-conscious world, it is appropriate to ask: is cool-chain technology
always necessary? Can energy consumption be reduced by storing and transporting
produce at higher temperatures than that currently utilized by employing other tech-
nologies to inhibit ripening and senescence? One such technology is to reduce activ-
ity of ethylene or the concentration around produce or the receptivity of produce to
ethylene action. The following sections in this chapter will examine how technology
that either reduces exposure to ethylene, or inhibits ethylene action or synthesis, can
reduce or even eliminate the need for refrigeration.

8.3 EFFECT OF ETHYLENE CONCENTRATION

ON POSTHARVEST BEHAVIOR
For many years, a concentration of 0.1 μL/L was generally cited as the threshold
level for the activity of ethylene on postharvest behavior (Burg and Burg, 1962;
Knee et al., 1985). Establishment of such a threshold level was probably related as
much to the ability to measure very low ethylene levels than any empirical evidence.
Peacock (1972) examined the effect on the ripening of green bananas exposed to
short bursts of ethylene, and found that while ripening was not immediately initi-
ated, the green life was shortened by any exposure to ethylene. He advocated that for
bananas there was no threshold level for ethylene action, and any ethylene level will
advance ripening.
The existence of a much lower threshold level for ethylene action was exam-
ined in a series of papers by Wills and colleagues (Table 8.1). They determined the
Low Ethylene Technology in Non-Optimal Storage Temperatures 171

TABLE 8.1
Postharvest Life of Climacteric and Nonclimacteric Produce Continuously
Exposed to Ethylene Concentrations of <0.005–10 μL/L in Published Reports
by Wills and Colleagues
Produce Temp (°C) Quality Issue Reference

Climacteric
Avocado 20, 10, 0 Ripening, chilling (0) Wills and Gibbons (1998)
Banana 20 Ripening Wills et al. (1999a)
Mango 20 Ripening Wills et al. (2001)
Peach 20 Ripening Wills et al. (2001)
Custard apple 20, 14 Ripening Wills et al. (2001)
Kiwifruit 20, 0 Ripening Wills et al. (2001)
Tomato 20 Ripening Wills et al. (2001)

Nonclimacteric
Strawberry 20, 0 Rotting, senescence Wills and Kim (1995)
Lettuce 20, 0 Leaf tip browning Kim and Wills (1995)
Green bean 20, 5, 0 Yellowing (20), chilling (5 & 0) Wills and Kim (1996)
Pak choi, choi sum, gai lan 20, 0 Yellowing Wills and Wong (1996)
Chinese cabbage 20, 0 Yellowing Wills et al. (1999b)
Orange 2.5 Chilling Wills et al. (1999b)
Broccoli, parsley, chive 20, 5 Yellowing Wills et al. (1999b)
Fancy lettuces (13 types) 5 Leaf-tip browning Wills et al. (1999b)

postharvest life of a range of climacteric and nonclimacteric produce that were con-
tinuously exposed to air containing a logarithmic decrease in ethylene concentration
from 10 to 0.001 μL/L (actually <0.005 μL/L, their limit of detection) at ambient
and reduced temperatures. They found that for all produce examined, the posthar-
vest life was extended linearly with logarithmic decrease in ethylene regardless of
whether postharvest life was due to ripening, rotting, yellowing, browning, chilling-
related injury, or general senescence. Examples of this relationship with ethylene
concentration are illustrated in Figure 8.1 using data generated for chilling-related
injury of “Valencia” orange stored at 2.5°C and ripening of “Hayward” kiwifruit
stored at 20°C.
Pranamornkith et al. (2012) showed a similar relationship for “Hort 16A” kiwi-
fruit (Actinidia chinensis) that were stored at 1.5°C. Fruit-ripening, as determined
by a range of attributes, was progressively delayed as the ethylene concentration
in the atmosphere around fruit was decreased in a logarithmic manner from 1 to
0.001 μL/L. The lowest concentration of 0.001 μL/L was confirmed as the actual
concentration, using the ultra-sensitive laser-based detection system developed by
Harren and colleagues in the Netherlands (Cristescu et al., 2013).
While it can now be categorically stated that the threshold level for a physi-
ological effect of ethylene on horticultural produce is much lower than 0.1 μL/L,
it would seem that the actual threshold level cannot yet be categorized. Since the
172 Advances in Postharvest Fruit and Vegetable Technology

150
Orange at 2.5°C
125
Time to chilling injury (days)
100

75

50

25

0
0.001 0.01 0.1 1 10
Ethylene concentration (log10 scale)
20
Kiwifruit at 20°C

15
Time to ripen (days)

10

0
0.001 0.01 0.1 1 10
Ethylene concentration (log10 scale)

FIGURE 8.1  Effect of ethylene concentration on the postharvest life of oranges at 2.5°C and
kiwifruit at 20°C as determined by chilling-related injury and ripening, respectively. (Data
from Wills RBH et al. 1999b. Aust J Expt Agric 39: 221–224 for “Valencia” orange, and Wills
RBH et al. 2001. Aust J Expt Agric 41: 89–92 for “Hayward” kiwifruit.)

longest postharvest life in the above studies occurs with the lowest ethylene concen-
tration, the threshold level is likely to be lower than 0.001 μL/L. The difficulty in
accurately measuring such low ethylene levels is a barrier to elucidation—it is pos-
sible that ethylene may still have some activity at one molecule per cell. However,
for commercial situations where ethylene emissions are derived from a range of
sources, any reduction in the ethylene concentration in the atmosphere around pro-
duce will be beneficial in extending postharvest life. It is immaterial whether the
source of ethylene in the atmosphere is from the produce itself, or from exogenous
sources. Wills et al. (2000) determined the ethylene level in the atmosphere during
Low Ethylene Technology in Non-Optimal Storage Temperatures 173

marketing, and found that the air in wholesale markets and distribution centers
­contained on average 0.06 μL/L ethylene, but was lower in supermarket retail out-
lets at about 0.02 μL/L. However, the level of ethylene in all premises was well
above any threshold level, and hence there was some decrease in postharvest life
throughout the marketing chain.
The importance of “background” ethylene levels, even in laboratory trials,
is illustrated with the study by Bower et al. (2003) who stored Bartlett pears in a
range of ethylene concentrations, including a nominal “0 μL/L” treatment in input
air. However, on analysis, the ethylene concentration was found to be 0.10 µL/L in
chambers of fruit stored at 1°C, and 0.32 µL/L in chambers at 2.5°C. The increase
was attributed to endogenous ethylene emissions from the fruit. This also raises the
importance of measuring the actual level of ethylene in containers in studies pur-
porting to examine the effect of ethylene on produce. While numerous studies have
been reported on the effect of ethylene, many have accepted that the input level of
ethylene is the actual level in the produce container. The efficacy of such findings
must be considered as suspect.

8.4 INTERACTION OF ETHYLENE AND TEMPERATURE

ON POSTHARVEST BEHAVIOR
Exposure of horticultural produce to low temperature and reduced ethylene
concentrations independently result in extensions to postharvest life. There are,
however, few published studies that quantify the interaction of temperature and
ethylene over the ranges likely to be encountered in commercial situations. The
most comprehensive study was reported by Wills et al. (2014) on inhibition of the
ripening of green bananas. They determined the green life (i.e., time to ripen) of
Australian “Cavendish” bananas at all combinations of ethylene at 0.001 (actual
<0.002), 0.01, 0.1, and 1.0 μL/L, and temperatures of 15°C, 20°C, and 25°C. The
relationship between green life and ethylene and temperature is given in Figure 8.2
and, as expected, shows that green life increased as the temperature and ethylene
concentration were reduced, with the greatest increases occurring at the lower end
of both the ethylene and temperature range. The value in the data is that it gives
various combinations of ethylene concentration and temperature that generate the
same green life. Thus, for any specified period required for transport or storage, it
is possible to manage the marketing scenario to retain produce quality but mini-
mize cost. Wills et al. (2014) also used the regression equation generated by the
data to determine the ethylene concentrations required to allow bananas shipped
commercially (by road within Australia and by sea from Central America to south-
ern Europe) to be carried at ambient temperatures, that is, without the need for
refrigeration. They concluded that such transport was feasible, although the longer
international route may require the use of some other ancillary technology (to be
discussed in later sections).
Wills and Kim (1996) also reported a similar relationship for the postharvest
life of green beans, where the maximum storage life was achieved at <0.005 μL/L
ethylene of 29, 20, and 11 days at 0°C, 5°C, and 20°C, respectively. For each tem-
perature, the postharvest life increased by 2–3 times as the ethylene concentration
174 Advances in Postharvest Fruit and Vegetable Technology

35

30

25
Green life (days)

20

15

10 0.001

5 0.01

L)
L/
0.1

(µ
0

ne
15

le
hy
20 1

Et
25
Temperature (°C)

FIGURE 8.2  Interaction of ethylene concentration and storage temperature on green life of
“Cavendish” bananas. (Data from Wills RBH et al. 2014. Postharv Biol Technol 89: 7–10.)

was increased to 0.01, 0.1, 1, and 10 μL/L. They found that ethylene levels around
beans in commercial markets were in the range 0.17–1.17 μL/L. Other reported stud-
ies examined too few temperatures and ethylene concentrations to make meaningful
assessment of the ethylene/temperature interaction, but it would not seem unreason-
able to expect that holding many produce at low ethylene would give a similar post-
harvest life as at low temperatures in current commercial situations where ethylene
levels can readily accumulate.

8.5  METHODS FOR INHIBITING ETHYLENE ACTION

Understanding the metabolism of ethylene has led to the development of various
methods to control ripening and senescence of fruits and vegetables. For example,
aminoethyoxyvinylglycine (AVG) and aminooxyacetic acid (AOA) inhibit ACC syn-
thase and, therefore, block the conversion of SAM to ACC. They act by reducing
ethylene production and are, therefore, ineffective against the action of exogenous
ethylene. While they have useful effects with some ornamentals, their development
with fruits and vegetables was effectively terminated due to potential toxic effects
on humans (Abeles et al., 1992). Other compounds that have some effects on ethyl-
ene production or action, but have not found any commercial application, include
3,5-diiodo-4-hydroxybenzoic acid (DIHB) (Robert et  al., 1975), ethylene oxide
(Lieberman et al., 1964), free radical scavengers such as benzoate and propyl gal-
late (Apelbaum et al., 1989), and polyamines such as spermidine (Wang, 1987), and
2,4-dinitrophenol (Yu et al., 1980).
Low Ethylene Technology in Non-Optimal Storage Temperatures 175

Considerable research has focused on the binding of ethylene to a specific enzyme

receptor to form an active complex that triggers the postharvest changes. The bind-
ing of ethylene to receptors is considered to take place reversibly at a site containing
copper, and the affinity of the receptor for ethylene is increased by the presence of
oxygen and decreased by carbon dioxide (Yang, 1985; Kanellis et al., 1989). Ethylene
action can then be modified by interfering with the binding of ethylene to receptors
or changing the quantity of receptors.
Blocking the metal receptor for ethylene was utilized by Beyer (1976) who
reported that a foliar spray of silver nitrate prevented a wide range of ethylene-induced
responses, including growth inhibition, abscission, and senescence in a range of
intact plants. Commercial use of silver as the thiosulphate salt was introduced for a
wide range of ornamentals to extend vase life. However, while treatment of fruit and
vegetables with silver ion has been shown to inhibit the action of ethylene, silver is
not a feasible option due to its toxicity. Even the use of silver on ornamentals is being
gradually restricted due to environmental concerns relating to disposal of treatment
solution, and of treated plant material at the end of the postharvest life.
Various cyclic olefins have been found to interact with the ethylene receptors
and, thereby, block tissue response to ethylene. Among them, 2,5-norbornadiene was
found to inhibit binding to receptor sites (Sisler et al., 1986), but the trans-cyclooc-
tenes were more effective (Sisler et al., 1990). However, both have an unpleasant odor
and rapidly diffuse from the receptor, which severely limits commercial use (Sisler
and Serek, 1999).

8.5.1  1-Methylcyclopropene
In recent years, the postharvest research community has focused on the cyclopro-
penes, which were recognized by Sisler and colleagues at North Carolina State
University, and, in particular, with 1-methylcyclopropene (1-MCP). The first publi-
cation on 1-MCP was by Serek et al. (1994), who showed that 1-MCP inhibited the
ethylene-induced abscission and wilting in potted flowering plants following a rela-
tively short prestorage treatment at nL/L concentrations for a few hours. Numerous
studies have since shown a beneficial effect of 1-MCP on a wide range of fruits and
vegetables, and it is now in commercial use in many countries for various produce,
with apples being the major beneficiary. The effectiveness of 1-MCP is considered
due to a physical similarity to ethylene that allows 1-MCP to bind to the metal in the
ethylene receptor where it remains bound for a long period.
While 1-MCP is an important consideration for this chapter, which is focus-
ing on low ethylene technologies that will allow produce to be stored above opti-
mal cool-chain temperatures, a comprehensive review of 1-MCP is discussed in
Chapter 6. Suffice it in this chapter to restate that common effects of 1-MCP include
a reduction in ethylene production, respiration, volatile synthesis, rate of softening,
loss of acidity, and loss of chlorophyll. These are typical of delayed ripening and
senescence that can be effected by inhibiting ethylene action. The list of effects
can also include a reduced incidence of various physiological disorders of apples
and chilling-related injury of various fruits, which would also seem to be related to
interference with the action of ethylene. However, 1-MCP is not a universal panacea,
176 Advances in Postharvest Fruit and Vegetable Technology

as issues on uneven ripening can occur in produce such as bananas (Golding et al.,
1998; Harris et al., 2000).
The commercial success of 1-MCP can be attributed in large part to the existence
of patent protection and the absence of any prior manufacture of 1-MCP. The current
patent-holder, Rohm and Hass (Spring House, PA), through its subsidiary, Agrofresh,
markets 1-MCP under the trade name SmartFresh™. This provided the funding
needed to support international research to demonstrate the effects of 1-MCP, con-
duct toxicological studies and obtain registration for usage in many countries. In the
current complex regulatory environment and community resistance to new chemi-
cals, the lack of patent protection is a significant impediment to commercialization
of any new chemical based technology.
Of direct relevance to this chapter is a commercial trial reported by McCormick
et al. (2012), in which “Gala” apples treated with 1-MCP were successfully stored at
a higher temperature than a room of untreated fruit. 1-MCP was applied at the cur-
rent commercial rate in Germany (0.625 μL/L for 24 h), then stored in a controlled
atmosphere room (2% CO2, 1.6% O2), but the temperature was maintained at 4°C,
which was 2.5°C higher than the control room. Fruit removed from storage at vari-
ous times and evaluated for quality after seven days at ambient temperature to simu-
late marketing showed that 1-MCP-treated fruit at the higher storage temperature
were preferred by a consumer sensory panel. An energy audit after six and eight
months showed a 35% reduction in energy was achieved by storage at the higher
temperature. Thus, a more acceptable product was marketed, but with considerable
energy saving.

8.5.2 Nitric Oxide
Nitric oxide (NO) is a highly reactive gaseous free radical that, traditionally, was
considered as an industrial byproduct toxic to plants and animals. However, exten-
sive research over the last 20 years has found NO to be metabolized by mammals,
and having a regulatory role in many human physiological systems. This has led to
the therapeutic use of NO for various conditions, although high levels result in det-
rimental effects.
The existence of NO in higher plants was first reported by Leshem and Haramaty
(1996) who found emissions from pea seedlings. Of particular interest to posthar-
vest horticulture was their finding of an inverse relationship between emitted NO
and ethylene, and that the addition of an NO-releasing compound decreased ethyl-
ene production. The link between ethylene and NO was furthered by Leshem et al.
(1998) who reported that an NO-releasing compound reduced the rate of ethylene
production and delayed postharvest changes in a range of climacteric and nonclimac-
teric produce. In addition, endogenous NO levels were higher in unripe fruits than
in ripe and in freshly-cut, compared to senescing flowers. Thus, endogenous NO and
ethylene emissions were inversely related and the addition of NO counteracted the
senescence-promoting effect of ethylene.
Considerable research on the postharvest effects of NO has been conducted in
many countries over the last 10 years, and the findings from these studies have
been reviewed in Chapter 9. A summary of the information, presented in Chapter 9,
Low Ethylene Technology in Non-Optimal Storage Temperatures 177

shows that exogenous NO, by fumigation or dipping in an NO-donor solution, inhib-

its ripening, senescence, chilling-related injury, and development of disease on a
wide range of intact and fresh-cut fruit and vegetables. In addition to inhibiting the
production of ethylene, NO has been demonstrated to reduce respiration, ion leak-
age and oxidative stress, enhance the activity of a range of antioxidant enzymes,
and inhibit polyphenol oxidase (PPO) activity. The key factor through which NO
is affecting metabolism is not known, but inhibition of ethylene production and/or
action is a likely important component.
Postharvest application of NO is thus a potential new technology to reduce losses
of horticultural produce. NO appears to be synthesized by all plants and can, there-
fore, be considered as a naturally occurring substance. It would then be analogous to
ethylene which, while it has toxicity and handling issues, is an approved postharvest
treatment. The human synthesis of NO and its therapeutic use should also be positive
in securing regulatory approval. However, plants and mammals do not have totally
identical pathways for metabolism of NO and it will need to be proved that NO in
fruit and vegetables does not generate toxic byproducts such as nitrosamines. An
impediment to commercialization of an NO treatment is the lack of current patent
protection. Some organization needs to expend considerable time and resources to
develop the safety and efficacy information to gain regulatory approval in many
countries, but lack of exclusivity for the treatment would be a major deterrent to
commercial operators.
Use of NO gas would require gas-tight fumigation chambers and the monitor-
ing of atmospheric discharges. A more convenient delivery system of a solid that
degrades to release NO gas in the presence of produce, as proposed by Wills et al.
(2007b), offers greater ease of application. Only a few of the many solid NO-donor
compounds have been evaluated, but, currently, sodium nitroprusside (SNP) seems
to offer potential for commercial use due to its stability in water and a long history
of safe clinical use. A new potentially enticing treatment option is to apply a non-
NO-containing compound that stimulates endogenous NO production. The limited
research on this aspect has revealed a divergent group of benign compounds. This
could be particularly appealing for regulatory purposes if the compound was either
naturally occurring or had GRAS or similar other safe status for foods.

8.5.3 Nitrous Oxide
Nitrous oxide (N2O) is a naturally occurring atmospheric gas mostly originating
from soil through the action of aerobic bacteria (Anderson and Levine, 1986). It is
chemically inert and has had extensive use for anesthesia in dentistry as it is gener-
ally considered to be nontoxic with an exposure limit of up to 50 µL/L over a pro-
longed period (Quarnstrom, 2013). It has a linear structure similar to carbon dioxide
with which it has similar physical properties, including high solubility in aqueous
media. There are no reports of endogenous nitrous oxide in plants.
Interest in the postharvest effects of nitrous oxide was initiated by the Air Liquide
laboratories in France, and Fath et al. (1990) filed a patent for treating horticultural
produce with nitrous oxide to extend postharvest life based on its antiethylene prop-
erties. Published data by the Air Liquide group (Gouble et al., 1995) showed that
178 Advances in Postharvest Fruit and Vegetable Technology

nitrous oxide delayed the ripening of tomato and avocado fruits by extending the
lag period before ethylene synthesis increased, and lowering the ethylene produc-
tion rate. The level of nitrous oxide used was quite high at 80% as the effect was
achieved by essentially displacing nitrogen from air atmosphere while retaining 20%
oxygen. The mode of action was considered to be similar to carbon dioxide, but as
nitrous oxide is not toxic to tissues, a higher concentration could be safely main-
tained around produce and presumably induce a stronger effect. Since the nitrous
oxide atmosphere was maintained around produce throughout storage, the technol-
ogy is limited to controlled-atmosphere storage situations.
Short-term exposure to nitrous oxide was reported by Benkeblia et al. (2001) and
Benkeblia and Varoquaux (2003). They found that for onions there was a limited
response that required high levels of nitrous oxide up to 100% for 4–15 days, with
a reduced incidence of rotting during subsequent storage but there was no effect
on the incidence of sprouting. At the end of the exposure period there was a small
transient reduction in respiration and a similar increase in soluble solids and acid-
ity. Qadir and Hashinaga (2001) examined short-term exposure of two, four, and
six days to 80% nitrous oxide in 20% oxygen followed by ambient air storage at
20°C, and found inhibited development of postharvest decay in apple, guava, per-
simmon, mandarin, strawberry, and tomato that had been inoculated with seven
fungal species. They also prestored strawberries in 10%–80% nitrous oxide with
20% oxygen for two, four, and six days, followed by ambient storage at 2°C, and
found nitrous oxide reduced the incidence of B. cinerea with the magnitude of the
effect being dose- and time-dependent. Thus the most effective treatment was 80%
nitrous oxide for six days. Lichanporn and Techavuthiporn (2013) treated longkong
fruit with 90% nitrous oxide vapor for 3 h, followed by storage at 13°C and 90%
RH, and found delayed development of pericarp browning with a higher level of
phenolic compounds but lower levels of the browning associated enzymes, phenyl-
alanine ammonia lyase, polyphenoloxidase, and peroxidase. Even though the effect
of nitrous oxide is considered to be similar to carbon dioxide, there have not been
any published studies comparing the effects of the two gases.

8.6 METHODS FOR REMOVING ETHYLENE

FROM THE ATMOSPHERE
8.6.1  Ventilation
The simplest technology to reduce the concentration of ethylene in the atmosphere
around horticultural produce in an enclosed space is to pass ambient air through
the container. Ambient air in rural areas contains a low level of ethylene at about
0.001 μL/L (Alberta Environment, 2003). While the trend in modern research is to
seek high-technology solutions to postharvest problems, the value of simple systems
should not be overlooked. Ventilation is an old physical method but has the potential
to be resurrected as a low-cost, environmentally friendly technology.
Ventilating storage chambers with external ambient air was one of the earliest
postharvest technologies utilized, although its primary function was for tempera-
ture maintenance in cave- or cellar-storage. Cooler external air was drawn into the
Low Ethylene Technology in Non-Optimal Storage Temperatures 179

chamber to maintain a low temperature, but it would have provided a bonus benefit
through reducing the accumulation of ethylene. The technology often relied on con-
vection, where the more dense cool external air flowed down into the chamber and
the hotter chamber air rose out of the chamber. The technique is still used in less-
developed regions with a cold winter, and where refrigerated facilities are expensive
and not widely available.
The ventilated transport of bananas by rail in Australia was advanced by the
designs of E.W. Hicks, a physicist at the Division of Food Preservation and Transport
of CSIRO (Commonwealth Scientific and Industrial Research Organisaton; the
Division is now the CSIRO Division of Animal, Food and Health Sciences) (Hicks
and Holmes, 1935). The standard covered-rail-van was modified by inclusion of lou-
vered panels into the van walls (Figure 8.3). Forward movement of the van drew
external air across boxes of bananas in the van. This minimized the warming of
bananas and also prevented the accumulation of ethylene. The overnight transport
of bananas from tropical and subtropical growing areas some 1000 km to temperate
markets was in such vans until the 1960s when they were gradually superseded by
refrigerated road transport, which was perceived to be more flexible in operation but
are now a considerable cost to the industry.
The efficient application of ventilation to a commercial situation requires knowl-
edge of the rate of ethylene production at the temperature of the storage chamber

FIGURE 8.3  One type of louvered rail vans that was used for long distance transport of
bananas in Australia.
180 Advances in Postharvest Fruit and Vegetable Technology

or that encountered during a transport route, the desired storage and/or transport
period, and the required ethylene concentration that will allow produce to retain
acceptable quality during the postharvest period. Such a study was conducted by
Wills et al. (2012) who modeled the effect of rates of ventilation on removal of a con-
tinuously generated ethylene supply from a chamber that simulated a stack of banana
packages. The modeling also examined the effect of ventilation on water loss from
fruit, as weight loss is an undesirable byproduct of excessive ventilation. Their data
indicated that ventilation with ambient air in conjunction with a small water tank
coupled to inexpensive computer-controlled data loggers on road vehicles can be a
cost-effective method to prevent ripening of bananas during long-distance transport.
Ventilation could be a valuable technology in developing countries as a reliable, low-
cost technology for fixed storage situations where refrigeration is either not available
or the energy cost is prohibitive.

8.6.2  Absorption and Oxidation

Ethylene is chemically quite reactive with a range of compounds able to react with the
double bond. Early attempts to reduce ethylene levels in the atmosphere around produce
was by adsorption onto activated charcoal and variants such as brominated charcoal,
as well as a range of diatomaceous earths, clays, and minerals. Most of these did not
bring the desired beneficial effect on produce due to insufficient ethylene adsorption
capacity and/or extent of reactivity of the adsorbed ethylene. Reid and Dodge (1995)
evaluated a number of adsorption products and found limited reduction in ethylene,
with the exception of potassium permanganate (discussed in Section 8.6.2.1). There
are, however, a number of recently released commercial products that do not disclose
the mode of action or the material used, but they appear to act by adsorption—their
efficacy is yet to be determined independently of the manufacturer or marketing agents.
In recent years, a range of scrubber units have been developed that use metal cata-
lysts to improve the efficiency of carbon-based reactivity. For example, Martínez-
Romero et  al. (2009) improved reactivity by incorporating 1% palladium into
activated carbon and regenerated its activity by heat pulsing. Such devices are suit-
able for storage rooms rather than individual packages.

8.6.2.1  Potassium Permanganate

Potassium permanganate (KMnO4) is a strong oxidizing agent that reacts with ethyl-
ene and generates carbon dioxide and water as the main end-products. It has a long
history of human use as a mild antiseptic to purify water or disinfect skin lesions.
An early use with vegetables was as a disinfectant while washing (in a weak solution
of potassium permanganate). Its first reported use to reduce the ethylene concentra-
tion in the atmosphere around postharvest produce was by Forsyth et al. (1967), on
apples. Subsequent studies have reported that the inclusion of potassium perman-
ganate decreased the ethylene concentration and delayed the ripening of a range of
climacteric produce.
Of particular interest is the research program in Australia led by Kevin Scott on the
ambient storage of bananas in sealed polyethylene bags. The initial report by Scott and
Roberts (1966) found that bananas in sealed bags generated a modified atmosphere
Low Ethylene Technology in Non-Optimal Storage Temperatures 181

and resulted in a delay in ripening. The low-oxygen and high-carbon dioxide atmo-
sphere reduces the sensitivity of fruit to ethylene (Liu, 1976), so a much higher eth-
ylene level will be tolerated before ripening is induced. The subsequent study (Scott
et al., 1970) included potassium permanganate that had been adsorbed onto an inert
porous solid (vermiculite), which reduced the ethylene concentration inside the sealed
bag and further delayed ripening; fruit ripened after 21 days, compared to 14 days in
a sealed bag without ethylene absorbent and seven days for air-stored fruit. A series
of semi-commercial trials between Australia and New Zealand (Scott et al., 1971a,b)
showed all treated fruit arrived in the international market still unripe.
Scott and Gandanegara (1974) examined the storage of green bananas over the
temperature range of 10–37.5°C in sealed polyethylene bags, with and without inclu-
sion of potassium permanganate, and found that the time to ripen at all temperatures
was extended by a modified atmosphere with a further extension due to reduction in
ethylene from the absorbent. Of relevance to the storage of produce in a nonrefriger-
ated environment, the study found that in the presence of potassium permanganate
the time to ripen at 25°C was 38 days and at 30°C it was 28 days. Their data suggests
that it would be feasible for sealed-bag transport of bananas with ethylene removal
to cope with any of the current world trading routes from tropical to temperate coun-
tries, to be conducted without refrigeration.
The ability of potassium permanganate to oxidize ethylene, and thereby delay
ripening, has been extended to a range of other climacteric fruit such as avocado
(Hatton and Reeder, 1972), mango (Esguerra et al., 1978), and kiwifruit (Scott et al.,
1984). Fewer studies have been conducted with nonclimacteric produce, but retarda-
tion in loss of green color and rotting in lemons (Wild et al., 1976) and lettuce (Kim
and Wills, 1995) has been shown, while Kim and Wills (1998) have demonstrated
inhibited rotting in strawberries.
Potassium permanganate is now commercially available in pellet form from a
diverse range of manufacturers in many countries, although the major market
appears to be for removal of odors from air-conditioning systems in buildings. It
would seem that a barrier to better commercial uptake is the lack of definitive studies
determining the actual amount of potassium permanganate to keep ethylene at a suf-
ficiently low concentration to maintain quality in individual produce for the desired
marketing period under specific storage conditions. Wills and Warton (2004), work-
ing with a laboratory prepared product on alumina beads, showed that temperature,
humidity, and the amount of absorbent all have an impact on the efficiency of eth-
ylene removal. They also found that the efficiency of ethylene removal had declined
to 10% of incoming ethylene when close to 50% of the original potassium perman-
ganate was still present in the alumina beads. They made calculations from known
rates of ethylene produced by various produce, and showed that at 20°C and 90%
RH, the amount of absorbent required in a unit package was manageable, but such
use was questionable for produce with higher levels of ethylene production or for
very long storage periods.

8.6.2.2  Ozone and Active Oxygen

Ozone is well known to the food industry as a strong oxidizing agent that can remove
pathogens and a range of undesirable contaminants from plant and animal products.
182 Advances in Postharvest Fruit and Vegetable Technology

However, in such systems, the level of ozone in the atmosphere must be controlled,
as it has toxic properties in plants and animals and a level of 0.1 μL is a common
permitted limit of exposure in the workplace.
The reaction of ethylene and ozone is well-documented (Dickson et al., 1992).
The first reported use of ozone on postharvest produce was reported by Gane
(1935) who claimed success in reducing ethylene in the atmosphere of chambers
of bananas, but found it difficult to control the level of ozone, which caused severe
fruit injury. Scott et  al. (1971a,b) used a UV lamp that emits mainly at 254 nm
but with trace emissions at 185 nm among other wavelengths and found rapid loss
of ethylene in the atmosphere around bananas in a sealed chamber but without
any fruit injury. They speculated that while ozone was produced at 185 nm, it
was rapidly degraded to atomic oxygen, which was more effective in oxidizing
ethylene than ozone. Scott and Wills (1973) then developed a system in which
the atmosphere from a chamber was circulated through a scrubber containing
UV lamps emitting at the required ratio of 185 and 254 nm where ethylene was
degraded but ozone did not emerged from the scrubber. Absoger of France (2013)
now manufactures an ethylene scrubber that uses a catalyst of titanium oxide that
is activated by a UV lamp to react ethylene with oxygen at elevated temperature.
A heat-exchanger is incorporated to allow the heat to be removed before the air is
returned to the storage chamber.
A range of ozone generators are commercially available and Skog and Chu (2001)
evaluated one such unit in a storage room and found that ozone at a concentration of
0.04 μL/L prevented the accumulation of ethylene in apple and pear storage rooms.
They also found that mushrooms, broccoli, and cucumbers could tolerate this ozone
level without damage. They suggested that a potential use for ozone generators is in
wholesale storage rooms where ethylene-producing produce such as apples are cos-
tored with ethylene-sensitive produce. Similarly, Lavigne et al. (2008) reported on
a study conducted by the United States army in conjunction with equipment manu-
facturer Primaira, to find alternate cost-effective methods for storing fruit and veg-
etables on military vessels and camps in a low ethylene environment. They found
an ozone generator developed by Primaira was able to maintain a low ethylene level
at a cost that was acceptable. The commercially available ozone generating systems
come in a range of sizes and prices, and would need to be assessed for individual
situations for cost and technical efficacy.

8.6.2.3  Prestorage Modified Atmospheres

An interesting alternative to maintaining a modified atmosphere during storage or
transport is the use of a short-term exposure to a low-oxygen or high-carbon dioxide
atmosphere followed by storage in ambient air. Wills et al. (1982) showed that hold-
ing green bananas in reduced oxygen for three days delayed ripening by about 40%
compared to air stored fruit—the ripening times being 19 and 14 days, respectively.
The authors suggested that the primary effect was due to inhibition of some aspect
of endogenous ethylene production, as the internal ethylene concentration was lower
following low-oxygen treatment, and ripening in all treatments coincided with rapid
increase in ethylene production. There was, however, no added benefit when carbon
dioxide was added into the atmosphere. Wills et al. (1990) examined bananas from
Low Ethylene Technology in Non-Optimal Storage Temperatures 183

more than 20 plantations over three seasons and confirmed that a three-day premar-
keting holding in nitrogen delayed ripening by about 40%. However, some injury
occurred on 4% of fruit but this developed on areas with existing mechanical dam-
age. Klieber et al. (2002) examined shorter exposure periods but found no increase
in green life for bananas stored in nitrogen for up to 24 h at 22°C. It would, therefore,
seem that a three-day exposure was required to modify the ethylene metabolic system.
There have been a range of other reports of either prestorage in low oxygen or
high carbon dioxide, extending postharvest life through decreased respiration or
better maintenance of appearance. For example, Couey and Olsen (1975) stored
“Golden Delicious” apples in high carbon dioxide (10%–25%) for 7–14 days and
obtained better retention of quality during subsequent storage in air, and Couey and
Wright (1975) delayed ripening of “d’Anjou” pears after prestorage in >10% CO2
followed by air or controlled atmosphere storage. Wills et al. (1979) found that the
respiration at 20°C of carrot, potato tubers, and zucchini was reduced by about 40%
after exposure to either high carbon dioxide (>10%) or low oxygen (<2%) for a few
days, and a reduced respiration was maintained for at least two weeks when returned
to storage in air. While these studies did not examine the effect on ethylene, it is sus-
pected that inhibition of ethylene synthesis could be at least a contributing factor to
the extended postharvest life in subsequent ambient air storage. However, regardless
of the mechanism involved, short-term holding in a low-oxygen or high-carbon diox-
ide atmosphere does offer a nonrefrigerated technology to extend postharvest life.

8.7  ENVIRONMENTAL BALANCE

While the storage of horticultural produce at a reduced temperature is very effective
at extending the postharvest life through delay in ripening, senescence, and micro-
bial growth, temperature control through refrigeration comes at an increasing cost
and greenhouse gas emissions. An important effect of reducing the storage tem-
perature is to reduce the endogenous production of ethylene, and render produce
less sensitive to ethylene, so that a higher concentration is required for the same
physiological action. It is then axiomatic that reducing the concentration of ethylene
around produce will also generate an extension in postharvest life, and could replace
or reduce the need for refrigeration.
There is, however, a general lack of appreciation by industry of the benefit to be
derived from reducing the ethylene level around produce. This is in large part due
to the relatively small number of studies that include ethylene concentrations well
below the previously perceived threshold level of 0.1 μL/L, and which accurately
monitor the actual ethylene level in treatments during storage. Published reports that
include ethylene levels <0.01 μL/L all show maximum postharvest life was attained
at the lowest concentration examined. This implies that a greater postharvest life
would be achieved if produce were exposed to even lower ethylene concentrations.
The limitation in most studies is the limit of detection of ethylene, which, for gas
chromatography, is 0.002–0.005 μL/L, while 0.001 μL/L can be detected by pho-
toacoustic laser-based detection systems. It can be speculated that ethylene con-
centrations of <0.001 μL/L may give larger extensions in postharvest life but are
logistically difficult to attain with any certainty.
184 Advances in Postharvest Fruit and Vegetable Technology

Wills et  al. (2000) developed a rating scale for nonclimacteric produce stored
at 20°C (that is, without refrigeration) based on 0.005 μL/L as the lowest possible
ethylene concentration that can be achieved, and hence was designated as retaining
100% of possible postharvest life. From calculations of published data on posthar-
vest life and ethylene concentrations for a range of produce (see Table 8.1), a loss of
10% was ascribed as the limit for an acceptable postharvest environment while 30%
was the limit for an unacceptable environment. The ethylene levels associated with
<10% loss was ≤0.015 μL/L with >30% loss occurring in >0.1 μL/L, which is the
previously considered threshold level. Wills and Warton (2001) attempted to extend
the rating scale to climacteric fruits, but concluded that a rating scale would need
to segregate individual produce into categories of low, medium, and high ethylene
sensitivity with each category having a different set of ethylene standards. 
To maintain produce quality throughout any specific marketing situation by con-
trolling the ethylene concentration, rather than through refrigeration, the question
then becomes what method of ethylene control should be utilized. The technology
used must not only be effective in maintaining quality but also needs to be less
costly than the system being replaced. Thus a cost-benefit analysis needs to define
a positive economic outcome. McCormick et al. (2012), in their assessment of stor-
ing “Gala” apples treated with 1-MCP at 2.5°C higher than untreated fruit, showed
that the 1-MCP-treated fruit were more acceptable to consumers and delivered a
35% saving in energy. While 1-MCP is a relatively low-cost option, there is a lim-
ited range of produce that can be successfully treated with 1-MCP. Numerous fruit
have been shown to subsequently exhibit abnormal ripening, or cannot be easily
ripened on demand. Use of nitric oxide is analogous to 1-MCP with a short prestor-
age treatment at a low concentration. A potential advantage of nitric oxide is that it
is naturally produced by plants and animals. However, there is a range of technical
and regulatory hurdles that need to be overcome before commercialization on foods
becomes possible (these issues are discussed in Chapter 9).
The most environmentally friendly method to minimize the accumulation of eth-
ylene around fruit and vegetables is by ventilation with atmospheric air—no synthetic
chemicals are needed, and the air is free. Ventilation can be achieved during transport
by directing the natural movement or air across a load of produce or in room or con-
tainer storage by the inclusion of a fan. The ability to manage water loss is the major
challenge but, with modern sensors, computer chips, and small heat exchangers, this
should be manageable. For large storage rooms, the use of active oxygen scrubbers
is similarly environmentally friendly as excess ozone or active oxygen are degraded
before the ethylene-free air is returned to the storage chamber. However, they come at
some cost, which needs to be considered in a cost-benefit evaluation.
The use of ethylene absorbents such as potassium permanganate is a relatively
low-cost option that is suitable for individual packages or containers. Disposal of
the absorbent on arrival at the market destination obviates the need for returnability,
an issue that needs to be resolved for more sophisticated technology. While ethyl-
ene absorbents are effective in nonsealed packages, they are most efficient when
included in a sealed modified atmosphere unit. The system then gains the benefit of
reduced ethylene, and inhibition of metabolism from the reduced oxygen and ele-
vated carbon dioxide.
Low Ethylene Technology in Non-Optimal Storage Temperatures 185

There are, thus, a range of technologies that can reduce the ethylene concentra-
tion in the atmosphere around produce, or inhibit the action of ethylene and, thereby,
extend postharvest life. For produce with a relatively short marketing chain, the
increased postharvest life may be sufficient to store and transport without refrigera-
tion. For longer duration postharvest situations, the reduced ethylene would allow
storage at a higher temperature. It should be possible for most produce to use a tech-
nology that provides the required postharvest life at a reduced cost, and with some
reduction in greenhouse gas emissions.
Finally, the obsession with cool-chain technology has undoubtedly led to its use
in situations where produce are held for short periods, and marginal benefit, if any, is
obtained. An example could be in supermarket refrigerated display areas. Wills et al.
(2000) found the ethylene level in such areas to be quite low at 0.02 μL/L and, when
combined with the high rate of produce turnover, it is suggested that there would be
minimal change in produce quality if display cabinets were maintained at ambient
temperature.

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 9 Potential of Nitric
Oxide as a Postharvest
Technology
Ron B.H. Wills

CONTENTS
9.1 Rise of NO from Villain to Hero................................................................... 191
9.2 Postharvest Application of NO...................................................................... 194
9.2.1 Fumigation with NO Gas................................................................... 194
9.2.2 Dipping in Solutions of NO-Donor Compounds............................... 195
9.3 Effects of NO on Postharvest Life................................................................. 195
9.3.1 Effects on Climacteric Fruit.............................................................. 196
9.3.2 Effects on Nonclimacteric Fruit and Vegetables............................... 198
9.3.3 Effects on Fresh-Cut Produce............................................................200
9.4 Mode of Action of NO on Postharvest Produce............................................202
9.5 Potential for Commercial Usage of NO.........................................................205
Acknowledgment....................................................................................................206
References...............................................................................................................206

9.1  RISE OF NO FROM VILLAIN TO HERO

Nitric oxide (NO) is a colorless, highly reactive free radical gas. Its production as
an industrial byproduct or vehicle pollutant has long been associated with harmful
effects on animals, plants, and the environment. However, NO is also synthesized
naturally with major sources being through soil microbial activity and lightning
discharges, with a meaningful proportion from burning vegetation, which includes
discharges from cigarette smoke (Leshem 2000). An important rapid reaction of
NO is with atmospheric oxygen and ozone. The reaction has adverse environmental
effects through reducing ozone in the stratosphere and through the condensation of
the resulting nitrogen oxides to generate acid rain (Casiday and Frey 1998).
Interest in NO was revolutionized when NO was identified as the endothelium–
derived relaxing factor that caused dilation of human arteries and was thus impli-
cated in controlling blood pressure and blood distribution. As with many scientific
breakthroughs, there is considerable conjecture as to who should be credited with
the discovery. Robert Furchgott, Louis Ignarro, and Ferad Murad working indepen-
dently at universities in New York, California and Texas in the United States pub-
lished papers within a short period in 1987–1988 and were subsequently recognized

191
192 Advances in Postharvest Fruit and Vegetable Technology

as codiscoverers when the trio were awarded the 1998 Nobel Prize for Physiology
or Medicine “for their discoveries concerning nitric oxide as a signaling molecule
in the cardiovascular system” (Nobel Prize 2013). However, many eminent scientists
were convinced that a British team led by Salvador Moncada, who reported the same
finding in Nature in 1987 (Palmer et al. 1987) should have been included with the
Nobel recipients (Berrazuela de 1999).
Extensive research over the last 25 years has found NO to be metabolized in
mammals by nitric oxide synthase (NOS, EC 1.14.13.39) which catalyzes the oxida-
tion of the amino acid, l-arginine to l-citrulline with the release of NO (Figure 9.1).
It is now known to have a regulatory role in many human physiological systems and
is used as a therapeutic adjunct in the treatment of a range of medical conditions. The
multiple actions of NO in mammals arise from its ability to readily diffuse through
cell membranes into hydrophilic regions of the cell, such as the cytoplasm, as well
as the lipid phase of membranes (Tuteja et al. 2004). NO has been identified as a sig-
naling molecule for functions such as stimulating host defenses in the immune sys-
tem, regulating development of arteriosclerosis and regulating neutral transmission
in the brain (Jaffrey and Snyder 1995; Lloyd-Jones and Bloch 1996; Bogdan 2001;
Karpuzoglu and Ahmed 2006). NO is also active in peripheral nervous systems,
including the respiratory tract, the digestive system, the urinary tract and cerebral
vasculature (Feldman et al. 1993; Rand and Li 1995).
Probably the most publicly recognized action of NO is its involvement as a vaso-
dilator in the process of penile erection. In the early 1990s, researchers at Pfizer in
the UK developed a new drug called UK-92,480 to treat angina by relaxing coronary
arteries. Early trials showed it was not very effective at easing angina but many
of the men reported more frequent and longer-lasting erections. In 1988, the com-
pound, sildenafil citrate, marketed as Viagra, was released and, as they say, the rest is

CA**
H 2N N H H2N O

C C

NH Calmodulin NH

CH2 CH2
Nitric oxide + N O
CH2 synthase CH2

CH2 CH2

CH O2 CH
NADPH NADP
H2N COOH H2N COOH
L-arginine L-citruline

FIGURE 9.1  Biosynthesis of NO. (Reprinted from Leshem YY, Wills RBH. 1998. Biologia
Plantarum 41: 1–10. With permission.)
Potential of Nitric Oxide as a Postharvest Technology 193

history. Viagra works by inhibiting an enzyme that reduces the dilating effect of NO
on penile arteries, thus prolonging an erection (Fisch and Braun 2005; Viagra 2013).
The effect of NO on plants was first studied in the 1970s but in the context of
whether NO-polluted air had deleterious effects on plant growth. Anderson and
Mansfield (1979) reported that NO can either enhance or reduce growth of tomato
plants, depending on the concentration of NO, but the critical NO concentration var-
ied between cultivars and levels of soil nutrients. Neighbour et al. (1990) proposed
that ambient NO was a determining factor of ozone toxicity in plants, as leaf injury
caused by ozone only occurred when NO was added at >0.002 μL ⋅ L−1. They also
suggested that NO at >0.10 μL ⋅ L−1 might inhibit the generation of stress ethylene.
The foundation for numerous recent studies on the potential benefit of NO
action on postharvest commodities can be attributed to the late Ya’acov Leshem
from Bar-Ilan University in Israel. Leshem was aware of medical interest in the
action and therapeutic benefits of NO and sought to determine if NO had a role
in the metabolism of higher plants. His first report (Leshem and Haramaty 1996)
on pea seedlings examined a role for NO in plant signaling. They added NO in
the form of the NO-donor compound S-nitroso-N-acetylpenicillamine (SNAP) to
pea leaves and found a greater emission of NO than ethylene and that both NO
and ethylene emissions increased when the ethylene precursor 1-aminocyclopro-
pane-1-carboxylic acid (ACC) was added. This led to the suggestion that NO could
regulate ethylene production in growing plants. Leshem et al. (1998) then reported
an inverse relationship between endogenous NO and ethylene released from four
unripe and ripe fruits (Table 9.1) and two cut flowers, while Leshem and Pinchasov
(2000) found that increased ethylene production, during ripening of banana and
avocado, coincided with reduced NO emission. Leshem et  al. (1998) also found
alfalfa sprouts that were heat stressed at 37°C showed increased production of
NO and decreased production of ethylene, suggesting a stress-coping role for NO.
Establishment of the inverse link between endogenous NO and ethylene was the

TABLE 9.1
Endogenous Concentration of NO in Unripe and
Ripe Fruit Flesh
NO Emission
(μM ⋅ h−1 ⋅ g−1 Fresh Wt)
Fruit Unripe Ripe
Avocado 8.4 0.8
Banana 17.5 3.6
Cherry tomato 72.0 34.5
Persimmon 12.6 4.2
Kiwifruit 6.8 4.2
Orange 4.9 2.4

Source: Adapted from Leshem YY, Wills RBH, Ku VVV. 1998. Plant
Physiol Biochem 36: 825–833.
194 Advances in Postharvest Fruit and Vegetable Technology

trigger for substantial research over the last 15 years on the effect of exogenous NO
to extend postharvest life to intact produce and fresh-cut horticultural produce and
to understand its mode of action.

9.2  POSTHARVEST APPLICATION OF NO

The application of NO to horticultural produce presents a number of technical and
logistical problems. These stem from NO, first, as a gas at operating temperatures,
and second, as a highly reactive free radical. A major consideration is the rapid
oxidation of NO by atmospheric oxygen, with Snyder (1992) reporting a half-life
of NO in ambient air of 5 s. This is exacerbated at the sub-ambient temperatures at
which horticultural produce are commonly stored, as the rate of oxidation of NO
increases with decreasing temperature due to increased intermolecular interaction
between NO and oxygen molecules (Tsukahara et al. 1999). Notwithstanding this,
the commercial availability of NO gas cylinders makes NO gas fumigation a poten-
tial method of application, at least for research.
However, postharvest treatment by dipping in aqueous solutions is a more com-
mon and preferred practise in the horticultural industry. The requirements for any
treatment are for the NO-generating compound to be quite stable as a solid, rela-
tively stable in solution but to quantitatively release NO when absorbed by pro-
duce. Due to the medical use of NO, a wide range of NO-donor compounds have
been developed that are relatively stable in solution. Hou et  al. (1999) reported
that NO-donor compounds can be classified into six categories based on the atom
or molecule attached to the NO releasing moiety. These categories are C–NO, N–
NO, O–NO, S–NO, h­ eterocyclic-NO, and transition metal-NO compounds. The
more important classes for biological purposes are N–NO, S–NO, and transition
metal-NO compounds (Hou et al. 1999). While it is not known which NO-donor
compounds would be approved for food use, many would seem to be safe as they
have been developed for therapeutic purposes but they are certainly useful for
research purposes.

9.2.1 Fumigation with NO Gas

The reactivity of NO in air led the early studies to treat horticultural produce with
gaseous NO in a nitrogen atmosphere, but the risk of anerobic metabolism at low
oxygen limited the treatment time to relatively short periods. However, Soegiarto
et  al. (2003) examined the rate of loss of NO gas at the relatively low concentra-
tions used to treat produce from atmospheres containing varying levels of oxygen.
They found that for 30–40 μL ⋅ L−1 NO, the rate of loss of NO was much lower than
expected (Snyder 1992) with the half-life of NO being about 4–6 h in 21% O2. The
rate of loss of NO from the atmosphere was shown to be much greater in the pres-
ence of horticultural produce than from air only, indicating a rapid uptake of NO by
produce (Table 9.2). They concluded that NO was sufficiently stable at the concentra-
tions and fumigation times utilized and the uptake by produce sufficiently rapid to
allow produce to be treated in normal air.
Potential of Nitric Oxide as a Postharvest Technology 195

TABLE 9.2
Half-Life of 40 μL ⋅ L−1 NO Gas in the Presence
of Horticultural Produce
Produce Half-Life (h)
Carnation 1.7
Strawberry 2.8
Banana 2.2
Air only 6.1

Source: Data from Soegiarto L. et  al. 2003. Postharv Biol

Technol 28: 327–331. With permission.

9.2.2 Dipping in Solutions of NO-Donor Compounds

While a wide range of relatively stable NO-donor compounds have been synthesized,
only a few have been used in postharvest horticulture research. The most commonly
NO-donor compound utilized is sodium nitroprusside (SNP) (Na2 [Fe (CN)5 NO]
2H2O). SNP is a transition metal NO complex class of donor compound that releases
the NO+ cation and which has found clinical use (Pitkanen et al. 1999; Wang et al.
2002). SNP is soluble in water and relatively stable in solution, resisting oxidation at
a neutral or slightly acidic pH (Verner 1974) and is widely available and relatively
inexpensive.
2,2′-(hydroxynitrosohydrazino)-bisethanamine (diethylenetriamine nitric oxide
(DETANO)) was applied by Noritake et al. (1996) to growing potato plants while
the first postharvest application of DETANO was by Bowyer et  al. (2003) on cut
carnation flowers. DETANO is claimed to be the most stable diazeniumdiolate with
a half-life in solution of 20 h at 37°C and pH 7.4 and to follow first-order kinetics in
degrading to two molar equivalents of the NO• free radical with the reaction being
dependent on solution temperature and pH (Lemaire et al. 1999; Fitzhugh and Keefer
2000). The half-life of 0.1 mM DETANO solution markedly decreases at low pH
with Davies et al. (2001) reporting it to be 24 s in solutions buffered to pH 2.
The only other NO-donor compounds that have been utilized are: Piloty’s acid
(N-hydroxybenzenesulfonamide), N-tert-butyl-α-phenylnitrone (PBN) and 3-mor-
pholinosyl-nominine (Sin-1). Each has been examined in one postharvest study.

9.3  EFFECTS OF NO ON POSTHARVEST LIFE

The rationale for most postharvest studies has been to examine whether application
of NO as a gas or by dipping in a solution of NO-donor compound inhibited ripen-
ing or senescence with some studies focused on control of physiological disorders or
diseases. The initial study on NO relating to horticultural produce was by Noritake
et al. (1996) who applied a DETANO solution to growing potato plants and found
it induced accumulation of the phytoalexin rishitin in tubers. The result suggested a
role for NO in stimulating the defence mechanism in plants possibly in response to
196 Advances in Postharvest Fruit and Vegetable Technology

abiotic stress. The first postharvest report was by Leshem et al. (1998). The study was
essentially a preliminary examination of a range of produce to assess the potential of
NO gas to extend postharvest life. They reported treatment of strawberry, broccoli,
cucumber, Chinese broccoli, kiwifruit and mushroom with a single concentration of
NO gas in the range of 0.05–0.25 μmoL ⋅ L−1 in a nitrogen atmosphere for 2–16 h
followed by storage in air at 20°C and found a 70%–180% extension of postharvest
life compared to untreated produce.

9.3.1 Effects on Climacteric Fruit

Studies on a range of climacteric fruit have shown that fumigation with NO gas can
regulate ethylene biosynthesis, which is accepted as the trigger for initiation of rip-
ening. Sozzi et al. (2003) fumigated pears with 10 μL ⋅ L−1 NO gas in air for 2 h and
showed decreased ethylene production, while fumigation with 10 and 50 μL ⋅ L−1
NO for 12 h delayed yellowing of the skin but did not affect fruit softening. It was
concluded that NO had differential effects on pear-ripening and suggested that a
time × concentration effect existed for applied NO. Zhu et  al. (2006) found that
peaches treated with five and 10 μL ⋅ L−1 NO gas in air for 3 h delayed ripening, as
measured by the firmness of the fruit, through reduced ethylene biosynthesis. They
also reported that NO inhibited lipoxygenase (LOX) activity and suggested that the
decrease in LOX activity might be associated with the inhibition of ethylene biosyn-
thesis. Singh et al. (2009) reported that Japanese plums fumigated with NO in air had
a reduced rate of respiration and ethylene production during ripening at 21°C. They
further showed that NO-fumigation caused a 3–4 day delay in ripening as evidenced
by restricted skin-color changes and retarded softening. While Flores et al. (2008)
did not report on ethylene, they found that peaches fumigated with 5 μL ⋅ L−1 NO
gas in nitrogen for 4 h at 20°C had a reduced respiration rate. They further showed
NO treated fruit remained firmer after storage and the degree of electrolyte leakage
was also lower. In mango fruit, Zaharah and Singh (2011b) showed that fumigation
with 20 μL ⋅ L−1 NO in air for 2 h at 21°C inhibited the activity of 1-aminocyclopro-
pane-1-carboxylic acid synthase (ACS) and 1-aminocyclopropane-1-carboxylic acid
oxidase (ACO), which led to a reduced level of ACC. The consequent lower levels
of ethylene reduced the activity of fruit-softening enzymes polygalacturonase and
pectinesterase but increased the level of endo-1,4-d-glucanase in pulp tissue during
ripening and cool storage. Eum et al. (2009b) showed that the ripening of tomato
fruit was delayed by fumigation with NO. In addition, Deng et al. (2013) reported
that a preharvest spray of SNP onto Golden Delicious apple trees 14 days before har-
vest delayed postharvest ripening through increasing the level of NO, which delayed
the accumulation of ethylene.
Chilling injury is a major storage problem for many produce when stored below a
critical threshold low temperature. NO has been implicated in improving chilling tol-
erance. Singh et al. (2009) reported that Japanese plums fumigated with 10 μL ⋅ L−1
NO in air had reduced incidence of chilling injury during storage at 0°C for 6 weeks.
Zaharah and Singh (2011a) fumigated mangoes with 10–40 μL ⋅ L−1 NO gas in air
for 2 h and showed that NO treatment not only delayed ripening during storage at
5°C but also reduced the incidence of chilling injury. Zhao et al. (2011) showed that
Potential of Nitric Oxide as a Postharvest Technology 197

tomatoes dipped in 0.02 mM SNP solution showed reduced chilling injury by induc-
ing NO accumulation and expression of a C-repeat/dehydration-responsive element
(CRT/DRE)-binding factors (CBFs), which play an important role in cold-response
regulation. They showed SNP protects tomatoes from cold-related injury by inducing
expression of LeCBF1 and that NOS activity may be inducing the NO accumulation.
In regard to other low-temperature physiological disorders, Zhu et  al. (2010)
found that fumigation of peaches with 15 μL ⋅ L−1 NO with intermittent warming
cycles of one day at 25°C after 14 and 28 days at 5°C was effective in preventing
the low-­temperature disorder of mealiness. Liu LQ et al. (2012) studied the develop-
ment of core browning, a low temperature physiological disorder of Yali pear after
fumigation with NO gas in nitrogen at 25°C for 3 h followed by storage in air at 2°C.
Treatment with 20 μL ⋅ L −1 NO was most effective at suppressing core browning.
Metabolic effects included an elevated level of NO in the fruit’s core tissue and a
reduced PPO activity and total phenols content but higher levels of glutathione and
ascorbic acid.
Xu et al. (2012) demonstrated that endogenous NO plays a role in chilling injury
development. They showed that loquat fruit stored at 1°C induced the accumulation
of endogenous NO but pretreatment with the NO scavenger 2-(4-carboxyphenyl)-
4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) abolished endogenous NO
accumulation and enhanced chilling-injury symptoms. The action of NO in alleviat-
ing chilling-injury symptoms was ascribed to stimulating the antioxidant defence
systems in the fruit. A role for NO in regulating oxidative systems was also attributed
by Wu et al. (2012) who found that fumigation of Chinese bayberry (Myrica rubra)
fruits with 20 μL ⋅ L −1 NO gas in nitrogen for 2 h inhibited ethylene production, the
incidence of disease and delayed the decrease in firmness, total phenolics and 2,
2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity. They suggested
that NO might enhance the resistance of tissues to decay by maintaining the balance
between the formation and detoxification of reactive oxygen species (ROS). Flores
et al. (2008) also measured a range of antioxidant enzymes and systems in peaches
and showed that fumigation with NO seemed to have a beneficial effect on the oxi-
dation equilibrium and the antioxidant capacity. Zhu et  al. (2008) also found that
application of NO in solution to kiwifruit reduced the accumulation of malondialde-
hyde (MDA), superoxide and hydrogen peroxide, delayed the decrease in vitamins
C and E, maintained the content of soluble solids, inhibited the activity of LOX and
peroxidase (POD), and increased the activity of superoxide dismutase (SOD) and
catalase (CAT) during storage.
It is of some interest that Zhang et  al. (2013) found an effect when arginine,
the metabolic precursor of NO, was applied to tomatoes. They dipped tomatoes in
0.2 mM arginine at reduced pressure (35 kPa) for 0.5 min then stored the fruit at
2°C. Arginine reduced the incidence of chilling injury and enhanced NOS activity
and thus the endogenous NO level. They also reported an accumulation of the poly-
amines putrescine and proline arising from increased activity of the related catabolic
enzymes that are often linked to amelioration of chilling injury symptoms.
The use of SNP as the source of NO has been evaluated on a limited number of
climacteric fruits with similar findings obtained as with the application of NO gas.
In addition to the papers already cited in this section, Zhang et al. (2008) found that
198 Advances in Postharvest Fruit and Vegetable Technology

plums dipped in 1 mM SNP for 3 min had reduced flesh browning after storage at
2°C but there was no effect on PPO activity. Sis et al. (2012) showed that peaches
dipped in 1 mM SNP for 5 min showed an extended postharvest life with a reduced
ethylene production rate and increased firmness and antioxidant enzymes activity.
Lai et al. (2011) showed that a 1 mM SNP dip applied to tomatoes delayed ripening
and reduced the incidence of fungal pathogens such as Botrytis spp. They further
showed enhanced activity of antioxidant enzymes in NO-treated fruit during stor-
age. The conclusion was that NO suppressed ethylene biosynthesis, stimulated the
activity of antioxidant enzymes and regulated the expression of age-related genes.
Zheng et al. (2011) inhibited rotting by Botrytis cinerea in tomato fruit by application
of an extract obtained from B. cinerea mycelia. The effect was attributed to stimu-
lation of the plant defense systems with an increase in expression of pathogenesis-
related protein 1(PR-1) gene within 2 h followed by enhanced production of NO.
The action was considered due to enhanced NOS activity as application of a NOS
inhibitor, N-nitro-l-arginine, eliminated the beneficial effect of the Botrytis extract.
Application of a nitrate reductase (NR) inhibitor, sodium azide, (NaN3) did not affect
the activity of the elicitor. Activities of phenylalanine ammonia lyase (PAL), chitin-
ase, β-1,3-glucanase, and PPO were also increased by the elicitor treatment as was
total phenols content.

9.3.2 Effects on Nonclimacteric Fruit and Vegetables

The effect of NO gas on nonclimacteric produce was reported by Wills et al. (2000)
who examined strawberries stored at 20°C and 5°C in humidified air, which con-
tained 0.1 μL ⋅ L−1 ethylene to simulate the ethylene level that might exist in a storage
room or transport-container filled with produce. They found that fumigation with
NO gas in nitrogen resulted in an increase in postharvest life with the most effective
concentration being 5–10 μL ⋅ L−1 NO at both temperatures. Zhu and Zhou (2007)
dipped strawberries in 5 μmol ⋅ L−1 SNP in water for 2 h at 25°C and found inhibited
ethylene production, respiration rate, and reduced ACS activity which led to a lower
level of ACC. However, the storage quality of the fruit in this trial was not reported.
Soegiarto and Wills (2004) fumigated broccoli, green beans and bok choy
(Brassica chinensis) with NO gas in air for 2 h then stored produce at 20°C in air
containing 0.1 μL ⋅ L −1 ethylene. All produce showed an extension in postharvest
life through inhibition of yellowing but the optimum concentration of NO differed
markedly, being about 50 μL ⋅ L−1 for green bean and bok choy and 4000 μL ⋅ L−1 for
broccoli. Eum et al. (2009a) have also reported that broccoli florets fumigated with
1000 μL ⋅ L −1 NO in nitrogen for 5 h delayed the onset of yellowing.
Other studies that used NO gas includes that by Zhu et al. (2009) who found that
Chinese winter jujube fumigated with 20 μL ⋅ L−1 NO gas in nitrogen at 22°C for 3 h
inhibited PPO and PAL activity. They also showed that treatment with different NO
concentrations from a saturated solution diluted to less than 1 μmol ⋅ L −1 inhibited
in vitro activity of PPO and PAL in a dose-dependent manner. Dong J et al. (2012)
found that mushrooms fumigated with 10–30 μL ⋅ L−1 NO gas in air for 2 h had
increased antioxidant activity as determined by assays of reducing power, chelat-
ing effect on ferrous ions, scavenging effect on hydroxyl free radicals, and DPPH
Potential of Nitric Oxide as a Postharvest Technology 199

radical scavenging activity. Furthermore, they also reported that NO f­umigation

enhanced phenolic and flavonoid contents and stimulated activity of PAL and chal-
cone synthase.
Use of SNP on longan fruit was examined by Duan et al. (2007) who reported
that dipping in 1 mM SNP solution for 5 min inhibited pericarp browning and pulp
breakdown and reduced PPO activity and the level of MDA, a marker of lipid per-
oxidation (Mittler 2002), but SNP treatment maintained higher levels of total soluble
solids and ascorbic acid during storage. Liu L et al. (2012) dipped “Glorious” oranges
in 30–100 μmol ⋅ L−1 SNP for 10 min and reported a higher level of titratable acid-
ity, soluble protein, ascorbic acid and reducing sugar, a lower weight loss, and lower
soluble solids concentration. Ku et  al. (2000) reported that rate of water loss was
reduced by about 20% from eleven fruits and vegetables and five cut-flowers follow-
ing a 24 h exposure to NO gas in nitrogen but did not measure the effect beyond this
period.
The role of NO in improving the tolerance of nonclimacteric fruit against chill-
ing-related injury was examined by Yang et al. (2011). Cucumbers were fumigated
with 25 μL.L −1 NO for 12 h at 20°C then stored at 2°C for 15 days. NO reduced the
increases in membrane permeability and lipid peroxidation associated with chilling
injury and delayed the increases in superoxide anion (O2̄ ) and hydrogen peroxide
levels. The NO-treated fruit also exhibited higher activity of SOD, CAT, ascorbate
peroxidase (APX), POD and higher DPPH-radical scavenging activity. Dong JF
et al. (2012) applied a saccharide extract of an unnamed yeast species to cucumbers
stored at 4°C and found a reduced incidence of chilling-related injury along with
reduced MDA content and ion leakage. The effects were attributed to an associated
increase in endogenous NO level. Application of an NO scavenger and NOS inhibi-
tors diminished the rise in NO and suppressed the induced tolerance to cold. The
results suggest that NO had an endogenous role in maintaining tolerance to chilling
in cucumber fruit by improving the antioxidant defense system.
Yin et al. (2012) examined the effect of NO on wound-healing of sweet potato.
Root discs were dipped in SNP or the NO-scavenger cPTIO and stored in the dark at
28°C and 85% RH. 0.5 mM SNP solution generated optimum wound-healing but the
effect was reversed by treatment with cPTIO. Other beneficial effects of SNP treat-
ment were enhanced activity of PAL, SOD, CAT, APX, and glutathione reductase.
It also maintained higher levels of total phenolics and ascorbic acid and total anti-
oxidative capacity, but peroxide production and MDA accumulation were inhibited.
It was suggested that NO promotes wound-healing of sweet potato by improving
PAL activity and activating the plant antioxidative defense system.
The interaction of NO and modified atmospheres was examined by Soegiarto
and Wills (2006) with strawberries and iceberg lettuce. The effect of low O2 was
examined by fumigating with 10 and 100 μL ⋅ L −1 NO gas in air and in 2% and 5%
O2 in air followed by storage at 10°C for strawberries and 5°C for lettuce in the same
atmospheres. They found an increase in postharvest life due to NO and to storage in
low O2 but there was no additional increase in postharvest life when both treatments
were applied concurrently. The effect of elevated CO2 was examined by placing
produce in a sealed polyethylene bag and injecting NO gas at 10 and 20 μL ⋅ L −1
into the bag. The atmosphere stabilized after seven days at about 20% CO2–3%
200 Advances in Postharvest Fruit and Vegetable Technology

TABLE 9.3
Postharvest Life of Strawberries Held at 10°C in a Modified
Atmosphere Package with NO Gas Injected into the Package
Treatment Postharvest Life (Days)
Air 4.0
MAP 7.2
10 μL ⋅ L−1 NO in air 6.0
10 μL ⋅ L−1 NO in MAP 10.7

Source: Data from Soegiarto L, Wills RBH. 2006. Aust J Expt Agric 46: 1097–
1100. With permission.

O2 for strawberries and 5% CO2 –13% O2 for lettuce. The results showed that the
postharvest life was enhanced by NO fumigation and was further enhanced by
coapplication of the modified atmosphere, presumably due to the CO2 (Table 9.3).
Jiang et al. (2011) examined the effect of modified atmospheres and NO on button
mushrooms but used DETANO as the source of NO. They dipped mushrooms for
10 min in DETANO solution and stored produce in sealed modified atmosphere
biorientated polypropylene bags at 4°C for up to 16 days. Treatment with 1 mM
DETANO maintained a high level of firmness, delayed browning and cap-opening,
promoted the accumulation of phenols, ascorbic acid and reduced increases in both
respiration and hydrogen peroxide content. The DETANO dip also inhibited PPO
activity and increased activity of CAT, SOD, and APX throughout storage period.
However, they did not measure the effect of the treatments without modified atmo-
sphere packaging and hence it is not known whether the effects of the NO and MAP
treatments were additive.

9.3.3 Effects on Fresh-Cut Produce

Fresh-cut produce is a growing sector of the fresh market with growth driven by
consumer demand for ready-to-eat or ready-to-cook fruit and vegetables. However,
fresh-cuts are more perishable than intact produce due to stress inflicted during
preparation, which involves removal of protective epidermal cells and damage to
exposed cells on the cut surface. Apart from the added risk of contamination by plant
or human pathogens, shelf-life is compromised due to an increase in respiration,
ethylene evolution, water loss and enhanced surface browning (Watada et al. 1996;
Artes et al. 1998). Development of surface browning is of particular concern as its
appearance is a potent trigger for consumer rejection.
The effect of NO on the development of browning on the cut surfaces of apple
slices was examined by Pristijono et al. (2006) who fumigated “Granny Smith” apple
slices with NO gas in air. They showed a delay in the onset of browning on the apple
surface during storage at 0°C with the most effective treatment being 10 μL ⋅ L−1
NO for 1 h. They also reported that this treatment was effective in inhibiting sur-
face browning of “Royal Gala,” “Golden Delicious,” “Sundowner,” “Fuji,” and “Red
Potential of Nitric Oxide as a Postharvest Technology 201

Delicious” apple slices. Pristijono et al. (2008) examined the effect of DETANO on
browning and reported that dipping in 10 mg ⋅ L−1 DETANO in pH 6.5 phosphate
buffer for 1 min was the most effective treatment in inhibiting surface-browning
of apple slices stored at 0°C. They showed it was necessary to buffer the DETANO
solution to be slightly acidic due to vacuolar acids leaking into the dip solution and
degrading the DETANO before it was absorbed into the apple slice. They also found
that dipping in DETANO solution was more effective than fumigating with NO gas
in inhibiting browning. Huque et  al. (2013) reported that apple slices dipped in a
buffered DETANO solution and fumigated with NO gas had a lower level of total
phenols, inhibition of PPO activity, reduced ion leakage, and reduced rate of respira-
tion, but showed no significant effects on ethylene production or lipid peroxide level
as measured by MDA and hydrogen peroxide, with DETANO having a greater effect
than NO gas. In addition, they found that apple slices dipped in a chlorogenic acid
solution enhanced the development of browning but subsequent dipping in DETANO
solution negated the effect of chlorogenic acid while fumigation with NO gas gave
partial relief. Huque et al. (2013) suggested that an increase in phenols occurs on
the apple surface soon after cutting and the effectiveness of NO to inhibit surface-
browning may relate to minimizing the activity of phenols on the cut surface, pos-
sibly in conjunction with reduced PPO activity.
Huque et al. (2013) also found that the NO-donor compound, Piloty’s acid, inhib-
ited browning in apple with the optimum aqueous dip solution concentration being
100 mg ⋅ L −1. They compared the optimum concentrations of 500 mg ⋅ L−1 SNP and
100 mg ⋅ L −1 Piloty’s acid dissolved in water with 10 mg ⋅ L−1 DETANO in phos-
phate buffer and fumigation with NO gas to inhibit browning. While all forms of
NO extended the postharvest life of apple slices, the order of effectiveness was
DETANO > SNP > NO gas = Piloty’s acid.
The effect of NO gas and DETANO on iceberg lettuce slices was examined by
Wills et  al. (2008). They found that the development of browning on the cut sur-
face during storage at 0°C was inhibited, with the optimal treatments being dip-
ping in 500 mg ⋅ L−1 DETANO in water for 5 min and fumigation with 500 μL ⋅ L−1
NO for 1 h, with DETANO being more effective than NO gas. The use of a water
dip compared to the need for a buffered dip with apple slices was attributed to the
higher pH of lettuce tissue not degrading DETANO in solution. Huque et al. (2011)
extended the findings to four other types of fresh-cut lettuce (Lactuca sativa)—
”Green Oak,” “Green Coral,” “Baby Cos,” and “Butter” lettuces—with storage at
5°C. They reported that dipping in a 500 mg ⋅ L−1 solution of DETANO buffered to
pH 6.5 and an aqueous solution of SNP inhibited the development of browning to a
greater extent than fumigation with NO gas for 2 h. Use of SNP was considered to be
the most feasible commercial option due to the stability of SNP in unbuffered water.
Studies on other fresh-cut produce includes that by Zhu et al. (2009) who fumi-
gated preclimacteric peach slices with a 5 μM NO solution for 10 min followed by
storage at 10°C for 10 days. Treatment with NO suppressed the increased rate of leak-
age and thus maintained compartmentalization between the enzymes and their sub-
strates. They also reported that NO increased PAL activity and total phenol content,
and inhibited PPO and POD activity. Cheng et al. (2009) showed that mature green
banana slices treated with 5 mM SNP solution had reduced ethylene production,
202 Advances in Postharvest Fruit and Vegetable Technology

which was associated with reduced ACO activity and expression of the MA-ACO1
gene. Yang et al. (2010) showed that bamboo shoots dipped in 0.5 mM SNP for 1 h
had delayed onset of external browning during storage for 10 days at 10°C. SNP also
inhibited PPO, POD, and PAL activity and maintained total phenol content during
storage. Furthermore SNP treatment inhibited the synthesis of lignin and cellulose
and delayed tissue lignification.

9.4  MODE OF ACTION OF NO ON POSTHARVEST PRODUCE

Postharvest research with NO was stimulated by the findings of Leshem and
Haramaty (1996) on pea leaves that NO and ethylene had an antagonistic relation-
ship. The ability of NO to inhibit ethylene production through inhibition of ACS and
ACO, resulting in a reduced level of ACC, has now been demonstrated for a range
of climacteric and nonclimacteric produce and is generally accepted as one of the
important actions of NO in inhibiting ripening and delaying senescence. However,
Huque et al. (2013) did not find a reduction in ethylene production in the NO-induced
inhibition of browning of apple slices but they suggested the lack of effect of NO
was due to the apples being postclimacteric and thus having a substantial production
of ethylene. Nonetheless, the data would suggest that ethylene is either not a direct
causal factor in the development of surface browning of apples or NO can inhibit
browning through other modes of action.
A common finding of many studies is that the applied NO reduced the rate of res-
piration. This is consistent with findings of Millar and Day (1996) and Zottini et al.
(2002) that NO affects the function of mitochondria in plant cells and reduces cell
respiration by inhibiting the cytochrome pathway. NO can then be ascribed an anti-
senescent action which could arise from a general reduction in the rate of cellular
metabolism. Whether this occurs via inhibition of ethylene action or independently,
needs to be determined. Reduced ion leakage arising from application of NO has
been found in many studies and could be ascribed as a side benefit of better mainte-
nance of cellular integrity through reduced general metabolism.
The role of NO in reducing oxidative stress and its involvement in signaling in
growing plants (Besson-Bard et al. 2008) would seem to also apply to postharvest
tissue. Reduced lipid oxidation caused by ROS is suggested to be a major cause of
membrane deterioration in plant tissues (Mittler 2002). Reduced levels of biomark-
ers for lipid peroxidation, MDA and hydrogen peroxide, have been found for various
postharvest produce, while NO has also been found to stimulate the activity of a
range of antioxidant enzymes and enhance PAL activity, which has been implicated
in defense mechanisms. Indeed, the stimulation of the phytoalexin, rishitin in grow-
ing potato tubers by NO (Noritake et al. 1996) may be an NO-moderated response to
either abiotic stress or pathogenic invasion. It would seem that research into whether
postharvest NO application can stimulate phytoalexins in other tissues and thus elimi-
nate or reduce the need for chemical biocides to combat pests and diseases could be
a worthwhile avenue for study. It is noted that Lazar et al. (2008) in an in vitro study
found that NO gas inhibited spore germination, sporulation and mycelial growth of
three postharvest fungi, Aspergillus niger, Penicillium italicum and Monilinia fruc-
ticola. NO may thus have a dual role in reducing the spread of postharvest diseases.
Potential of Nitric Oxide as a Postharvest Technology 203

Inhibition of PPO activity by NO has been shown in numerous intact and fresh-
cut produce and was associated with reduced internal- and surface-browning. PPO
contains two copper ions in its active center and Zhu et al. (2009) speculated that NO
could react with the copper to form copper–nitrosyl complexes, which could change
the normal structure of the active site in PPO and thus reduce PPO activity. There
may be potential for application of NO with processed fruits and vegetables, where
browning of juices and purees during storage is a common problem and treatment
with NO to reduce PPO activity may enhance the shelf-life of the processed product.
The action of NO on the phenolic substrates, which are substrates for PPO activity,
is not clear as the various studies give conflicting data, with some showing a reduced
level of phenols while others show an enhanced level.
Alleviation of chilling-related injury by NO is a relatively recent expansion of
the mode of action of NO. Endogenous NO production is triggered in response to
chilling-stress itself (Xu et al. 2012) or by application of some defense elicitor such
as a yeast extract (Dong J et al. 2012; Dong JF et al. 2012). Similarly, application of
a fungal extract has been shown to inhibit rotting by stimulation of NO (Zheng et al.
2011). Thus, application of exogenous NO could, therefore, seem to be enhancing a
natural defense response to chilling-related injury and disease resistance.
The interaction of NO with modified atmospheres has not been well studied. The
only report where NO and a modified atmosphere were simultaneously applied with
appropriate air-control treatments (Soegiarto and Wills 2006) showed that exposure
to both NO and elevated CO2 gave an added extension in postharvest life of strawber-
ries and lettuce over that arising from the individual components, whereas exposure
to NO and reduced O2 gave no added benefit. The findings suggest that NO and
CO2 have a different mode of action while that of O2 and NO is similar and hence
not additive. The benefits of modified atmospheres are generally considered to be
through an inhibition of respiration via a feedback signal from the respiratory gases.
NO has been applied as a gas and as various NO-donor compounds but all forms
have been reported to give a beneficial effect in inhibiting postharvest changes that
lead to an extension in postharvest life. However, the different forms of NO degrade
to release different NO moieties. DETANO, Piloty’s acid and NO gas release the NO•
free radical and SNP releases the NO+ cation (Zamora et al. 1995; Hou et al. 1999;
Saavedra and Keefer 2002). It might be expected that each NO moiety has differ-
ent reactivity on produce metabolism, with the moiety more directly linked to the
key metabolic action(s) being more effective in inhibiting postharvest change. Since
all NO compounds produced some beneficial effect, it would seem likely that the
released NO moieties are interconvertible to some extent. Direct comparison of the
different forms of NO has only been conducted with fresh-cut apples and lettuces and
only in relation to the development of surface-browning. Conclusions drawn from
these studies in relation to ripening and general senescence of intact produce must,
therefore, be made with some caution. For apple slices, Huque et al. (2013) found
all four NO forms inhibited development of browning with DETANO > SNP > NO
gas = Piloty’s acid, while for the four lettuces evaluated, Huque et al. (2011) found
SNP = DETANO > NO gas. As an applied treatment, fumigation with NO gas is
therefore less effective than dipping in DETANO and SNP solutions. However, the
effect of the solvent must also be considered. In the above studies, DETANO was
204 Advances in Postharvest Fruit and Vegetable Technology

dissolved in phosphate buffer, and SNP and Piloty’s acid in water, while NO gas
uses no solvent. Since both phosphate buffer and water alone gave some inhibition of
browning, the relative effectiveness of NO gas and DETANO would be, respectively,
higher and lower than that found for the actual applied treatments. For the lettuces,
Huque et al. (2011) calculated the effectiveness of NO as a percentage of the respec-
tive control treatments as SNP > NO gas > DETANO.
While a range of effects of NO on the metabolism of horticultural produce have
been identified, it is not clear when NO is acting at the chemical, enzymic, or genetic
level. It seems quite possible that NO is active on a range of systems, which could
utilize all three modes of action. Also, what is—or are—the key action(s) that gen-
erates—or generate—supporting reactions that lead to delayed ripening and senes-
cence? Do these actions differ from those leading to disease-resistance and inhibition
of chilling-related injury? Determining the action of endogenous NO in plants would
seem to be more complicated than in mammals as, in addition to the synthesis by
NOS in mammals, NO in plants can be synthesized by nitrate reductase NR, EC
1.6.6.1-3) which primarily catalyzes the conversion of nitrate to nitrite but under
stress conditions can generate NO (Leshem 2000; Yamasaki and Sakihama 2000).
However, Zheng et al. (2011) found NOS to be active in NO generated for defense
against Botrytis rotting in tomatoes, but NR was not active.
Studies are beginning to examine the effects of NO at the genetic level. The inhi-
bition of rotting in tomato fruit using a B. cinerea extract reported by Zheng et al.
(2011) was attributed to stimulation of the plant defense systems through expres-
sion of the pathogenesis-related protein 1(PR-1) gene followed by enhanced NOS
activity and enhanced production of NO. Zhao et  al. (2011) demonstrated that an
elevated NO content up-regulated the expression of a cold-regulation gene (LeCBF1)
and also induced antioxidative processes to scavenge ROS resulting in treatment
improved tolerance to cold in tomatoes in cold storage while Eum et  al. (2009b)
showed that NO can regulate ripening in tomato fruit through genes that regulate
ethylene biosynthesis. Kuang et al. (2012) found NO enhanced the expression of the
histone deacetylase 2 gene, DlHD2, but suppressed the expression of two ethylene-
responsive genes, DlERF1 and DlERF2, of longan fruit during storage at 25°C. It
seems that NO may be acting on a range of systems, which have different levels of
importance in different produce and in response to differing postharvest stresses.
It is still an open question whether exogenous NO is acting as a foreign agent
similar to the inhibition of ethylene by 1-MCP or is accentuating the level—and
hence activity—of endogenous NO. Early studies by Leshem (2000) indicated that
the level of endogenous NO in mature produce is relatively low. Utilization of NO
scavengers as used by Xu et al. (2012) would seem to be useful to investigate these
scenarios. The interaction between applied ethylene and NO would appear worth
examining, given the central role of ethylene in ripening and general senescence.
Based on the early observations of Leshem (2000), it is not unreasonable to suggest
that NO has a major role in maintaining cellular integrity and organization during
development and maturation of the produce but is replaced by the deteriorative effect
of ethylene in mature produce, which have reached the end of their developmental
life. Application of exogenous NO could then be analogous to applying a juvenile
hormone to prevent aging.
Potential of Nitric Oxide as a Postharvest Technology 205

9.5  POTENTIAL FOR COMMERCIAL USAGE OF NO

Since NO seems to be synthesized by all plants it can be considered as a naturally
occurring substance. As such, it would be analogous to ethylene which, while it has
toxicity and handling issues, is an approved postharvest treatment. The human syn-
thesis of NO and the therapeutic use of various forms of NO should also be positive
factors in gaining regulatory approval. However, plants and mammals do not have
totally identical pathways for metabolism of NO. Of particular interest is the presence
in higher plants of NR which catalyzes the conversion of nitrate to nitrite. Nitrites
have the potential to be converted to nitrosamines which are potent carcinogens
(Walker 1990). The addition of NO gas or individual donor compounds would need
to be shown to not generate nitrosamines. The use of NO with ornamentals would
not pose such a regulatory hurdle and regulatory approval should be easier to obtain.
Use of NO gas would require gas-tight fumigation chambers and the monitoring
of atmospheric discharges from treatment chambers but these are not insurmount-
able issues, particularly if the NO was added to air rather than into a nitrogen-rich
atmosphere. A more convenient delivery system for NO gas could be along the lines
proposed by Wills et al. (2007) who developed a solid mixture that quantitatively
released NO gas in the presence of horticultural produce. The mixture of DETANO,
citric acid and wheat starch (added as a filler and moisture absorbent) in the ratio
of 1:10:20 was found to be stable when stored in dry air. However, in humid air,
absorption of moisture from the atmosphere led to reaction of DETANO with citric
acid and the evolution of NO gas (Figure 9.2). They found that when the dry mixture
was placed in containers of strawberries and mushrooms, the moisture given off by
produce activated the mixture and resulted in a similar extension in postharvest life
as achieved by direct fumigation with NO gas. The industrial use of such a solid
mixture could be through tablets or sachets placed in packages in a packing house
or even added into storage rooms. The package would not need to be sealed as the
steady release of NO gas over a few days is rapidly absorbed by the surrounding
produce.

80

60
NO (μL · L–1)

40

20

0
0 5 10 15 20 25
Time (days)

FIGURE 9.2  Atmospheric concentration of NO gas released from a solid mixture of

DETANO:citric acid:starch (1:10:20 w/w) held in a sealed container in air at 20°C and 95%
RH. (From Wills RBH, Soegiarto L, Bowyer MC. University of Newcastle, Australia, unpub-
lished data.)
206 Advances in Postharvest Fruit and Vegetable Technology

Only a few of the many NO-donor compounds have been evaluated and then on
only a few produce. To date, SNP would seem to offer potential for commercial use
due to its stability in water and resistance to oxidation at a neutral or slightly acidic
pH (Verner 1974). It has also had a long history of safe clinical use for hypertensive
and cardiac conditions (Ziesche and Franciosa 1977), which could assist in gaining
regulatory approval. While DETANO has been shown to be more effective than SNP
for some produce, it is unstable at even mildly acidic pH (Hrabie et al. 1993). The
quality of water used in commercial operations is variable and would require the use
of a buffered solution to ensure a near neutral pH is maintained throughout dipping.
For ornamentals, the addition of DETANO or some other solid NO-donor compound
to vase water would seem to offer a commercial opportunity.
An alternative treatment option which is even more under-explored is to apply
a non-NO containing compound that stimulates endogenous NO production. This
could be particularly appealing for regulatory purposes if the compound was either
naturally occurring or had GRAS or similar other safe status for foods. A poten-
tial research line could be a yeast extract as used by (Dong J et  al. 2012; Dong
JF et  al. 2012) to reduce chilling-related injury of cucumber. The recent paper of
Zhang et al. (2013), reporting that application of arginine, the precursor of the NOS
catalyzed synthesis of NO (Figure 9.1), stimulated NO production by tomatoes and
resulted in reduced chilling-related injury, offers an interesting simpler option. Since
l-­arginine is an essential amino acid, there should be minimal hurdles in gaining
regulatory approval. Another option is to apply some physical condition such as heat
or atmosphere that stimulates NO production. An early paper by Leshem et al. (1998)
showed that increased production of NO occurred when alfalfa sprouts were heat
stressed at 37°C.

ACKNOWLEDGMENT
Tribute is paid to the late Prof. Ya’acov Leshem, Bar-Ilan University, Israel–an
innovative biologist for whom scientific curiosity was the finest art. His ability for
cross-disciplinary thinking pioneered scientific investigation into the role of NO in
postharvest horticulture. The author was a beneficial recipient of Leshem’s will-
ingness to share his vision and values in the all too brief time spent in research
collaboration.

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 10 Methyl Jasmonate
in Postharvest
Ahmad Sattar Khan, Zora Singh, and Sajid Ali

CONTENTS
10.1 Introduction................................................................................................... 211
10.2 Biosynthesis of Jasmonates............................................................................ 212
10.2.1 Linolenic Acid and Lipases............................................................... 212
10.2.2 Lipoxygenase..................................................................................... 213
10.2.3 Allene Oxide Synthase...................................................................... 213
10.3 Methods of MJ Application........................................................................... 214
10.4 Interaction with Other Plant Growth Regulators........................................... 214
10.4.1 Ethylene............................................................................................. 214
10.4.2 Abscisic Acid..................................................................................... 215
10.4.3 Salicylic Acid..................................................................................... 215
10.4.4 Polyamines......................................................................................... 216
10.5 MJ and Fruit-Ripening.................................................................................. 217
10.6 MJ and Fruit Quality..................................................................................... 217
10.6.1 Color Development............................................................................ 218
10.6.2 Fruit-Softening.................................................................................. 218
10.6.3 Soluble Solids and Sugars.................................................................. 218
10.6.4 Phytochemicals.................................................................................. 226
10.6.5 Oxidative Stress................................................................................. 226
10.7 MJ and Physiological Disorders and Diseases.............................................. 226
10.7.1 Chilling-Related Injury...................................................................... 227
10.7.2 Inhibition to Disease.......................................................................... 227
References............................................................................................................... 228

10.1 INTRODUCTION
Jasmonic acid (JA) or its methyl ester (methyl jasmonate, MJ), collectively known as
jasmonates (JAs), are naturally occurring plant growth regulators (PGRs) (Sembder
and Parthier 1993; Creelman and Mullet 1995) that are involved in variety of
responses in higher plants including cell division, fruit growth (Kondo and Fukuda
2001), and fruit ripening (Fan et al. 1998b). The level of JAs has been reported to
change during fruit development. In apple, the concentration of endogenous MJ is
higher at maturity compared to the initial fruit-growing period (Kondo et al. 2000),

211
212 Advances in Postharvest Fruit and Vegetable Technology

whereas in strawberry (Gansser et al. 1997), grape (Kondo and Fukuda 2001), and

sweet cherry (Kondo et al. 2000), MJ concentration is higher at initial stages of fruit
growth followed by a steady decrease during fruit development.
MJ was discovered in floral extracts of Jasminium grandiflorum L. (Demole et al.
1962) while JA was isolated from the pathogenic fungus Lasiodiplodia theobromae
(Aldridge et al. 1971). However, it was not until 10 years later that its biological activ-
ity in extracts from Artemisia absinthium L. was reported (Ueda and Kato 1980).
JAs have now been discovered in numerous other species and are considered as ubiq-
uitous in nature (Hamberg and Gardner 1992; Meyer et al. 1984; Ueda et al. 19991).
Considerable research has been conducted on controlling the rate of ripening of
climacteric fruit after harvest. Conventional approaches suitable for the extension of
postharvest storage life of such fruit include use of cold storage, but this has limited
value with many fruit due to their sensitivity to chilling-related injury. An alterna-
tive method is to inhibit ethylene biosynthesis and/or prevent ethylene actions, and
this has been achieved with exogenous application of various compounds such as
1-methylcyclopropene (1-MCP) (Khan and Singh 2009), polyamines (PA) (Mattoo
and Handa 2008), 1-aminoethoxyvenylglycine (AVG) (Rath et al. 2006), and nitric
oxide (NO) (Zaharah and Singh 2013). However, many fruit show uneven ripen-
ing following application of these chemicals. Exogenous application of MJ has been
found to induce uniform fruit ripening in various climacteric fruit (Fan et al. 1998b);
this chapter reviews MJ/JA biosynthesis and actions, interaction of MJ/JA with other
PGRs, their role in fruit ripening and color development, and effect on physiological
disorders and diseases.

10.2  BIOSYNTHESIS OF JASMONATES

Biosynthesis of jasmonates is initiated by conversion of linolenic acid (LA) to
13-hydroperoxylinolenic acid (HLA) by lipoxygenase (LOX). HLA is converted by
hydroperoxide dehydrase or hydroperoxide dehydratase allene oxide synthase (AOS),
also known as allene oxide cyclase (AOC), to 12-oxo-phytodienoic acid (12-oxo-
PDA), the immediate precursor of JA. JA can be catabolized to form MJ and other
conjugates with biological activity (Hamberg and Gardner 1992). Accumulation of
JA in reaction to physical wounding, and/or treatment with some other elicitors and
systems can effectively be blocked with LOX inhibitors (Baldwin et al. 1996; Doares
et al. 1995; Farmer et al. 1994; Pena-Cortes et al. 1993).

10.2.1 Linolenic Acid and Lipases

Application of LA to fruit plants results in the accumulation of JA, which indicates
that the level, availability, and distribution of LA can determine the rate of syn-
thesis of JA (Farmer and Ryan 1992). However, the level of free LA in fruit may
increase manifold after wounding (Conconi et al. 1996), which partly results from
the release of LA from some phospholipids, and/or utilization of LA present in the
fruit tissues prior to wounding. The escalation in JA might result from the trigger-
ing of the phospholipases, which then releases LA from membranes (Farmer and
Methyl Jasmonate in Postharvest 213

Ryan 1992). Phospholipase D cleaves the head group of the phospholipids and trig-
gers the release of LA. Moreover, phospholipase D has also been reported to play a
significant role in plant defense mechanisms (Wang 1993). However, in some cases,
fatty acids may also be oxidized by LOX before release from lipids for synthesis to
JA. An oxidative burst associated with plant defenses may also stimulate the activ-
ity of phospholipases to release oxidized fatty acids for synthesis to JA (Bradley
et al. 1992). Observations that enzymes involved in JA biosynthetic pathways are
predominantly localized in the plastids (Bell et al. 1995). Blée and Joyard (1996)
suggested that some imperative mechanisms must be present to transport released
LA to the target plastids.

10.2.2 Lipoxygenase
Treatment with LOX inhibitors (Baldwin et al. 1996; Pena-Cortes et al. 1993) and/or
transgenic plants with the reduced activities of LOX (Bell et al. 1995) have reduced
capacity to synthesize JA. Plant LOX oxygenates LA at the position of 9 or 13, and
while 13-hydroperox-LA is generally the major product, the function of 9-hydroper-
oxides in the plants is imprecise (Ehret et al. 1994). Changes in the abundance and
distribution of LOX during development are due to expression of different LOX gene
family members. For instance, two LOX genes, “AtLox1” and “AtLox2,” (Bell and
Mullet 1993) have been identified. Because “AtLox1” lacks clear targeting sequences,
it is almost certainly localized in cytoplasm, while “AtLox2” is localized in chloro-
plasts (Bell et al. 1995). Plastid transit sequences suggest that LOX, which catalyzes
HLA, is restricted to chloroplasts (Peng et al. 1994).

10.2.3  Allene Oxide Synthase

Hydroperoxide lyase (HPLS) has been reported to cleave HLA to 6-carbon-alde-
hydes and 12-oxo-dodecenoic acid volatiles. JAs production requires that HLA must
be metabolized by AOS to produce allene oxide. AOS activity has been restricted
only to the outer envelope of plastids (Blée and Joyard 1996), but this needs to be
confirmed. Moreover, AOS-over-expression has been reported to increase the level
of JA. AOS activity has been found to be higher in tissues with higher JA levels, sig-
nifying that the differences in JA concentration, which occur during the development
phase of plants, may be caused by the variations in AOS-abundance and/or activ-
ity (Simpson and Gardner 1995). The enzymes catalyzing reduction of 12-oxo-PDA
and/or β-oxidation to JA have been validated in vitro but have not been extensively
characterized (Vick 1993). In tomato fruit, JL5 gene repressed the translation of the
HLA to 12-oxo-PDA (Howe et al. 1996), which, in turn, can be transformed in AOC
or AOS activity. An inhibitor of the biosynthesis of JA, diethyldithiocarbamic acid
(DIECA), was shown to reduce the levels of HLA to 13-hydroxy-LA (Feys et  al.
1994). This suggests that the DIECA inhibits the biosynthesis of JA by dropping the
precursor pools leading to allene oxide. Salicylic acid (SA), another facilitator of
some the plant defense reactions, impedes conversion of the 13-S-hydroperoxy-LA
to 12-oxo-PDA (Farmer et al. 1994; Pena-Cortes et al. 1995).
214 Advances in Postharvest Fruit and Vegetable Technology

10.3  METHODS OF MJ APPLICATION

Application of MJ can be done by either vapor or emulsions, but effectiveness var-
ies among cultivars, and the maturity of a produce (Ayala-Zavala et al. 2005; Buta
and Moline 1998; Gonzalez-Aguilar et  al. 2001, 2003; Meir et  al. 1996; Meyer
et al. 1991). Wang and Buta (1994) conducted the first application of MJ to alleviate
­chilling-related injury in Cucurbita pepo by dipping in an MJ emulsion, but vapor
treatment has been proved more effective for the prevention of chilling-related injury
in guava and papaya fruits (Gonzalez-Aguilar et al. 2003, 2004).
In general, vapor application of MJ is accomplished by soaking a filter paper in a
solution of MJ and placing the paper in a sealed container of fruit. The amount of MJ
applied in a treatment can be quantified by analysis of the headspace in the container
(Khan and Singh 2007). The required duration of exposure to MJ differs between
produce. For example, tomato fruit need to be treated with 10−5 M MJ for 3–4 h,
whereas mango requires exposure for 20–24 h at the same concentration (Gonzalez-
Aguilar et al. 2001). For fruit treated by immersion, the solution of MJ also needs
to include a surfactant at 0.05% concentration. The produce is then immersed in the
emulsion for a much shorter time of 1–3 min.

10.4  INTERACTION WITH OTHER PLANT GROWTH REGULATORS

MJ has been found to modulate expression of numerous genes that affect various
aspects of fruit development, some responses to abiotic and/or biotic stresses, and
regulation of ripening. It might be expected that MJ would interact with other PGRs.
The ubiquitin-mediated-protein dilapidation has been identified as a controlling
mechanism in numerous PGR pathways (Devoto et al. 2002). MJ signaling utilizes
this pathway with three MJ-signaling-genes, CORONATINE INSENSITIVE-1
(COI1), SGT1b/JAI4, and AUXIN RESISTANT-1 (AXR1), identified as part of
ubiquitin-proteasome-pathway. COI1 has a principal role in signaling of MJ and is
required for all MJ-dependent-responses (Feys et al. 1994; Xie et al. 1998). It has
also been found to encode F-box-protein and is the first signal for MJ in the ubiqui-
tin-mediated-protein breakdown (Xie et al. 1998). This hypothesis has further been
reinforced by demonstration that COI1 is found in an E3 type ubiquitin-ligase func-
tional complex (Devoto et al. 2002; Feng et al. 2003).
A target of COI1-dependent-proteasome degradation has been identified as RPD3b
histone deacetylase (Devoto et al. 2002). However, histone deacetylation is supposed
to lessen the accessibility of the chromatin to transcription machinery; therefore,
COI1-dependent-proteasome degradation of RPD3b may be a possible mechanism
for the MJ-dependent-transcription suppression (Lusser et  al. 2001). Conversely,
expression of RPD3b-associated histone deacetylase (RPD3a/HD19) has the reverse
effect, thus increasing the transcription of the MJ-induced ERF1-transcription fac-
tors and ERF1-targets (Zhou et al. 2005).

10.4.1 Ethylene
MJ and ethylene may either collaborate or act antagonistically in the regulation of
­various stress-related responses, such as disease or pathogen attack and wounding
Methyl Jasmonate in Postharvest 215

(Cuello et al. 1990; Devoto et al. 2002; Devoto and Turner 2003; Miszcak et al. 1995;
Farmer et al. 2003; Turner et al. 2002). In case of attack by pathogens, both MJ and eth-
ylene cooperate in a synergistic manner for activation of specific defense gene expres-
sion (Lorenzo et al. 2003; Xu et al. 1995). Fruit with inhibited synthesis of either MJ
or ethylene show reduced induction of defense and enhanced pathogen susceptibility
(Knoester et al. 1998; Staswick et al. 1998; Thomma et al. 1998; Vijayan et al. 1998).
However, for various wound responses, antagonistic interactions have been reported
between the MJ and ethylene for activation of reactions confined in damaged fruit tis-
sues (Rojo et al. 2003). Two MJ transcription factors, ERF1 and AtMYC2/JIN1, have
been found to participate in regulation of these defense interactions. ERF1 expression
is usually triggered by infection of necrotrophic pathogens and regulates in vivo expres-
sion of defense-related-genes PDF1.2 and b-CHI (Berrocal-Lobo and Molina 2004).
Moreover, expression of ERF1 and/or targets of ERF1 are prompted by MJ and ethyl-
ene, but this promotion further depends on synchronization of MJ and ethylene signal-
ing corridors. Numerous other ERFs are also prompted by MJ and ethylene interactions,
and are probably also involved in the regulation of the defense mechanism against
intruding pathogens, (Brown et al. 2003). Interestingly, ERF1 positively regulates the
expression of pathogen-response genes, and, ultimately, precludes the JA-mediated
induction of the wound-response gene VSP2 (Lorenzo et al. 2004). Two JA-regulated
transcription factors, JAMYC2 and JAMYC10, have been reported in tomato fruit and
found to specifically recognize a T/G box in promotion of the leucine aminopeptidase
(LAP) JA-induced gene (Boter et al. 2004). JAR1 genes encode an enzyme that has JA
adenylation activity, which forms conjugates among the JA and several other amino
acids, specifically with isoleucine (Staswick et al. 1992). JAR1 also conjugates JA with
the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC).

10.4.2  Abscisic Acid

Exogenous MJ inhibits fungal spore germination and shows synergistic interaction
with ABA, but this may not reflect endogenous functions (Staswick et  al. 1992).
Antagonistic interaction between MJ and ABA pathways have been perceived to
modulate the pathogen defense reactions, while, positive relations between MJ and
ABA have been reported in wound-responses (Anderson et  al. 2004). Similarly,
JIN1/AtMYC2 can be a major controller of interactions between MJ and ABA. In
addition to MJ, expression of AtMYC2 is also stimulated by ABA in a COI1-gene-
dependent way, which proposes that ABA leads MJ in AtMYC2-mediated activation
wound-responses (Lorenzo et al. 2004). Moreover, the deleterious effects of ABA
on MJ regulated-expression of defense genes (Anderson et al. 2004) might not be
caused merely by AtMYC2 activation by ABA. It would seem that ABA acts inde-
pendently of MJ in pathogen defense regulation and AtMYC2 is not be the solitary
point of convergence (Boter et al. 2004; Lorenzo et al. 2004).

10.4.3 Salicylic Acid
The relationship between salicylic acid (SA) and MJ has been intensively studied
(Devoto and Turner 2003; Li et al. 2004; Turner et  al. 2002). MPK4 that encodes
216 Advances in Postharvest Fruit and Vegetable Technology

MAP-kinase 4 is probably the major gene identified as a key regulator of the nega-
tive interaction between JA and SA in defense activation. It has been reported that
MPK4 is mandatory for induction of MJ-regulated defense genes, including PDF1.2
and THI2.1. MPK4 loss of the functions constitutively expresses SA-regulated genes,
possibly due to the elevated levels of SA. This suggests that a MAP-kinase-cascade
encompasses MPK4-regulated JA-SA-interaction by inhibiting biosynthesis of SA and
promotes perception of MJ. Likewise, constitutive ERF1 over-expression promotes
activation of the MJ and/or ethylene-dependent defenses and increases tolerance of
pathogens, which suggests possible positive interaction between MJ and ethylene, as
well as SA-signaling pathways (Berrocal-Lobo et al. 2002). It has also been reported
that induction of SA after infection of Pseudomonas inhibits accumulation of MJ with
this negative interface being controlled by NPR1 protein (Spoel et al. 2003). NPR1
stimulation by SA cooperates with participants of TGA transcription factors, and, ulti-
mately, triggers SA-dependent expression of PR genes. Surprisingly, negative effects
of NPR1 on the signaling of JA does not need its nuclear localization but appears to be
exercised via NPR1 function in the cytosol of fruit (Spoel et al. 2003). Moreover, NPR1
could not work solely, because the NPR1-related gene NPR4 has been reported to be
required for the full expression of MJ-induced PDF1.2 genes (Angelini et al. 2005).
MJ induces biosynthesis of anthocyanins by the induction of chalcone synthase
and dihydroflavonol-4-reductase gene expression (Saniewski et al. 1998). Therefore,
postharvest MJ application increased anthocyanin contents (Rudell et  al. 2002)
and the level of antioxidants in banana, guava, mango, and papaya (Kondo et  al.
2005). Application of MJ also enhances the activity of phenylalanine ammonia
lyase (PAL) and, consequently, the contents of various other flavonoids (Gonzalez-
Aguilar et al. 2004; Tomas-Barberan et al. 2001). Moreover, MJ promotes biosyn-
thesis of β-carotene in tomato (Saniewski and Czapski 1983; Saniewsky et al. 1997)
and apples (Perez et al. 1993). This effect may be due to direct stimulation of the
β-carotene or by indirect enhancement of β-carotene synthesis through promotion
of ethylene synthesis by MJ (Perez et al. 1993).

10.4.4 Polyamines
The polyamines (PA) mainly consist of putrescine, spermidine, and spermine
(Langebartels et  al. 1991), and play important roles in different characteristics
throughout plant development, including stress-responses and senescence (Angelini
et al. 2005). MJ treatment has been found to increase the levels of spermidine and
spermine but decrease putrescine (Wang and Buta 1994). Both spermidine and
spermine have been shown to be involved in lipid peroxidation reduction, degrada-
tive enzyme inhibition, and stabilization of the membrane structures (Escribano and
Merodio 1994; Smith 1985). The higher levels of PA in MJ-treated tissues appear to
be active in the reduction of chilling-related stress (Wang and Buta 1994).
Application of MJ to apple and plum fruit has been reported to increase the activi-
ties of the key ethylene synthesising enzymes, aminocyclopropane-1-carboxylic acid
synthase (ACS), and ACC oxidase (ACO), and hence stimulates synthesis of ethylene
(Khan and Singh 2007; Olias et al. 1992). Biosynthetic conduits for both ethylene
and PA share S-adenosyl methionine (SAM) as a metabolite and, hence, plant cells
Methyl Jasmonate in Postharvest 217

can commit SAM into synthesis of PA and/or ethylene (Cassol and Mattoo 2002).
Lower levels of MJ and JA have been reported in mango skin and pulp during early
growth stages, and increase during ripening, with ACC levels higher in pulp than
skin tissue (Lalel et al. 2003). This proposes that JA or MJ and ACC are related to
the ripening of mango.

10.5  MJ AND FRUIT-RIPENING

JA and MJ are usually present in low concentrations in different parts of plants, but
the highest levels are reported in fruit (Meyer et  al. 1984). In apples, endogenous
MJ has been found to be low during early fruit development but increase towards
harvest (Kondo et al. 2000). Similarly, Lalel et al. (2003) reported that the level of
MJ in mango pulp was significantly higher at harvest but decreased during ripen-
ing. On the other hand, levels of MJ in the nonclimacteric fruit such as strawberries
(Gansser et al. 1997; Mukkun and Singh 2009), cherries (Kondo et al. 2000), and
grapes (Kondo and Fukuda 2001), have been found to be higher at immature stages,
and decrease steadily during development. Furthermore, application of MJ to unripe
green strawberries enhanced respiration, production of ethylene, and synthesis of
anthocyanins, and chlorophyll degradation, thus suggesting a natural role for MJ in
strawberry-ripening (Perez et al. 1997).
Application of MJ triggers the ripening of climacteric fruit by increasing ethylene
production (Fan et al. 1997; Perez et al. 1997; Saniewski et al. 1987). MJ has been
found to also enhance development of aroma (Fan et al. 1997; Lalel et al. 2003; Olias
et al. 1992), and various pigment changes (Lalel et al. 2003; Perez et al. 1997, 1993).
A transient increase in endogenous MJ was observed in apple and tomato during the
onset of fruit-ripening, with promotion of ethylene synthesis (Czapski and Saniewski
1992; Fan et al. 1998a). JAs have been found to enhance ethylene production through
stimulation of ACS and ACO activity (Saniewski 1995).
MJ stimulates the production of ethylene, ACC, and ACO activity in the precli-
macteric stage of various fruit particularly apple (Fan et  al. 1997; Staswick et  al.
1992). Responses of fruit to exogenous MJ/JA, suggested that JAs may be involved
in the regulation of the early fruit development (Fan et al. 1997). Endogenous con-
centrations of MJ and JA increased during the commencement of ripening in apple
and tomato, increased rapidly prior to an increase in ethylene, and decreased during
the later stages of ripening (Fan et al. 1998a). Moreover, activities of ACS and ACO
have been reported to be stimulated by low concentrations of MJ (Khan and Singh
2007). Response of color changes to MJ vapor also depended on the fruit develop-
mental stage, and its affect was maximized as fruit began to generate ethylene (Fan
et al. 1998a). MJ treatment also caused a higher respiration and ethylene production
in white strawberries, while a decrease in carbon dioxide evolution and ethylene has
been observed in the ripe and overripe strawberry fruits (Perez et al. 1997).

10.6  MJ AND FRUIT QUALITY

Fruit quality consists of numerous attributes that encompass appearance, texture,
flavor, nutrient content, and phytochemicals (Schreiner and Huyskens-Keil 2006).
218 Advances in Postharvest Fruit and Vegetable Technology

Higher endogenous levels of MJ during fruit-ripening and exogenous application of

MJ have been reported in numerous studies to influence the postharvest fruit quality
parameters in both climacteric and nonclimacteric fruit, and these findings are sum-
marized in Table 10.1. Exogenous application of MJ has been found to improve fruit
color and increase aroma volatile production, chlorophyll degradation and carotene
synthesis, as well as enhance levels of antioxidants of many fruit along with reduced
incidence of rotting and chilling injury. Some of the studies reported in Table 10.1
on various effects of MJ on fruit quality are considered in more detail in the sections
that follow.

10.6.1 Color Development
A red peel color is an important quality parameter of many ripe fruit. Anthocyanins
are the major compounds responsible for red color in apples. MJ has been reported
to increase anthocyanin accumulation and, hence, peel color change in peach, straw-
berry, and apple fruits (Perez et  al. 1997; Saniewski et  al. 1998; Shafiq et  al. 2013;
Wang 1999). While this is generally considered due to enhanced synthesis of ethylene
by JAs (Fan et al. 1997; Saniewski and Czapski 1983), the effect for apples has been
suggested as being at least partly independent of ethylene (Fan and Mattheis 1999a;
Shafiq et  al. 2013). Moreover, increased sugar content in raspberries following MJ
treatment has been attributed to induced anthocyanin accumulation (Pirie and Mullins
1976). Similarly, ripe “Kent” mango that had been treated with MJ developed more
intense red and yellow colors and exhibited superior fruit quality compared to control
fruit (Gonzalez-Aguilar et al. 2001). Also, the natural peel-color change from green
to yellow developed faster and more uniformly in “Manila” mango exposed to MJ
(Herrera et al. 2004). Exogenous application of MJ also stimulated ethylene synthesis
and aroma volatiles in “Kensington Pride” mango during fruit-ripening. It would also
seem that pre- and post-harvest application of MJ can be used to improve fruit color
development at harvest and during postharvest handling in a wide range of fruit.

10.6.2 Fruit-Softening
MJ application slightly reduced the firmness in strawberries during storage (Perez
et  al. 1997), “Kent” mango exhibited a rapid loss of fruit firmness after storage
(Gonzalez-Aguilar et  al. 2001) and increased softening in Japanese plum fruit,
which was attributed to enhanced ethylene production and activity of ACS and ACO
enzymes (Khan and Singh 2007). In other studies, MJ did not significantly affect
fruit firmness during cold storage (Gonzalez-Aguilar et  al. 2000). When MJ was
applied in combination with modified atmosphere packaging (MAP) it resulted in
reduced loss of firmness in papaya fruit, but reduced firmness appeared to be related
to the effects of film-packaging rather than MJ (Gonzalez-Aguilar et al. 2003).

10.6.3 Soluble Solids and Sugars

MJ treatment affects the quality attributes such as soluble solids concentration (SSC),
titratable acidity (TA), and pH of a range of fruits. For example, MJ application to
TABLE 10.1
Effect of Exogenous Application of Methyl Jasmonate on Postharvest Quality of Fruit
Fruit Cultivar MJ Concentration Conditions Inferences Reference
Apple “Braeburn” 4480 mg/L 105 & 175 DAFB Increased red color, phenolic content, Ozturk et al. (2014)
and antioxidant capacity
“Delicious” 400 mg/L ethephon + 5 µM 12 h at 20°C Increase internal ethylene, firmness, Kondo et al. (2005)
1-MCP + 5 mM MJ malic acid, and aroma volatiles
“Cripps Pink” 10 mM Preharvest spray 169 Increased red blush, anthocyanins, Shafiq et al. (2013)
DAFB chlorogenic acid, phloridzin, and
flavanols
Methyl Jasmonate in Postharvest

“Cripps Pink” 1 or 5 mM Spray 3 week before Increased red blush, anthocyanins, Shafiq et al. (2011)
harvest and flavonoids
— 1000 ppm 20°C for 4 days Increased PAL activity, phenolics, Heredia and Cisneros-
and antioxidants Zevallos (2009)
“Fuji” 10 µM 1-MCP + 400 µM 20°C for 40 hr Increased anthocyanin and carotene; Rudell and Mattheis
MJ reduced chlorogenic acid (2008)
“Fuji” 2.24 and 4.48 g/L Preharvest spray 48 and Increased fruit size and color Rudell and Fellman
172 DAFB development; reduced fruit- (2005)
softening, and bitter pit
“Fuji” 10 mM 1-MCP + 2 mM 20°C for 15 days Inhibited ethylene production, Fan and Mattheis (1999b)
MJ alcohols, and esters
“Fuji” 2.24 g/L 120 h at 20°C Increased anthocyanin, carotene, and Rudell et al. (2002)
color development
“Golden Delicious” 0.75 µL 1-MCP + 2.0 mM 0°C for 14 week Increased production of esters Li et al. (2006)
MJ + 2 mM SA
“Golden Delicious” 400 mg/L ethephon + 5 µM 20°C for 12 h Increased internal ethylene, firmness, Kondo et al. (2005)
1-MCP + MJ malic acid, and aroma volatiles
(Continued)
219
 220

TABLE 10.1  (Continued)

Effect of Exogenous Application of Methyl Jasmonate on Postharvest Quality of Fruit
Fruit Cultivar MJ Concentration Conditions Inferences Reference
“Golden Delicious” 1 and 10 µM for 12 h 22°C till ripe Increased ethylene synthesis and Fan et al. (1998b)
improved color development
“Golden Delicious” 8 ppm Storage for 2 week Promoted β-carotene synthesis and Perez et al. (1993)
chlorophyll degradation
Avocado “Etinger”, “Fuerte” 2.5 µM 4–10 week at 2°C Reduced chilling-related injury and Meir et al. (1996)
“Hass” wound percentage
Banana “Namwa” 0.39 mM PDJ 6 and 12°C for 4 days Reduced chilling-related injury and Kondo et al. (2005)
increased antioxidative enzymes
“Cavendish” 0.1 mM 7°C for 5 days Increased chilling-tolerance over Zhao et al. (2013)
expression of ICE-CBF MaCBF1,
MaCBF2, MaCOR1, MaKIN2,
MaRD2, and MaRD5 genes
“Williams 8818” 1.5 mM Preharvest Higher chitinase, β-1,3-glucanase, Sun et al. (2013)
TPC, SOD, POD, PPO, CAT, and
PAL activities
Bayberry “Wumei” 10 μM 0°C for 12 days Reduced fruit decay, higher PAL Wang et al. (2009)
activity, increased TPC, flavonoids,
and anthocyanins
Blackberry “Chester Thornless”, 0.1 mM 4 week Higher TSS, flavonoids, antioxidants, Wang et al. (2008)
“Hull Thornless” anthocyanin, and TPC
Cherry tomato — 0.1% chitosan + 500 mM Incubated 7 days at Inhibited mycelial growth, reduced Chen et al. (2014)
MJ 28°C disease, higher activities of PPO,
POD, and PAL
Fragaria — 100 µM Stored for 9 days Increased ethylene, anthocyanin, Concha et al. (2013)
chiloensis expression of LOX, AOS, and
OPR-3 genes
Advances in Postharvest Fruit and Vegetable Technology
Grapefruit “Marsh seedless” 10 µM 4–10 week Reduced chilling-related injury and Meir et al. (1996)
wounds
Grape — 250 ppm  20°C for 4 days Increased PAL activity, phenolics, Heredia and Cisneros-
and antioxidants Zevallos (2009)
“Monastrell” 0.3 mM MJ + 10 mM Preharvest application Higher anthocyanin and flavonol Ruiz-Garcia et al. (2013)
BTH contents
Guava White and red flesh 10−4 and 10−5 M 5°C for 15 days, 20°C Reduced chilling-related injury & Gonzalez-Aguilar et al.
for 2 days ion leakage, increased sugars, PAL, (2004)
and LOX activities
Guava “Klomsalee” 0.39 mM PDJ Storage at 6°C and 12°C Reduced chilling-related injury and Kondo et al. (2005)
increased anti-oxidative enzymes
White variety 220 µM MJ + 1% CaCl2 6°C for 28 days Increased shelf-life Barriga-Tellez et al.
Methyl Jasmonate in Postharvest

(2011)
Loquat “Fuyang” 10 µM 20°C for 24 h, 1°C for Proline accumulation, reduced Cao et al. (2012)
35 days chilling-related injury, Δ1-pyrroline-
5-carboxylate synthetase, GABA,
ornithine δ-aminotransferase,
glutamate decarboxylase
“Fuyang” 24 µM 20°C for 24 h, 1°C for Reduced chilling-related injury, Cao et al. (2010)
35 days internal browning, increased
firmness, PAL, PPO, POD, and
polygalacturonase activities
“Fuyang” 10 µM 20°C for 24 h, 1°C for Reduced chilling-related injury, Cao et al. (2009)
35 days increased fruit firmness, and
activities of SOD and CAT
“Jiefangzhong” 10 µM 20°C for 6 days Reduced decay incidence and higher Cao et al. (2014)
polyamines
(Continued)
221
 222

TABLE 10.1  (Continued)

Effect of Exogenous Application of Methyl Jasmonate on Postharvest Quality of Fruit
Fruit Cultivar MJ Concentration Conditions Inferences Reference
“Jiefangzhong” 10 µM 20°C for 24 h, 1°C for Inhibited chilling-related injury, Cai et al. (2011)
35 days internal browning,
monodehydroascorbate reductase,
dehydroascorbate reductase, and
glutathione reductase activities
“Jiefangzhong” 10 µM 20°C for 6 days Reduced anthracnose rot and lower Cao et al. (2008)
disease incidence
Mandarin  “Ponkan” 100 µM 20°C for 4 days Inhibited disease incidence, higher Guo et al. (2014)
activities of PPO, POD, and CATs
Mango “Kensington Pride” 10−3 M 21°C Increased ethylene, skin color, fatty Lalel et al. (2003)
acids, total aroma volatiles
“Kent” 10−4 M 5°C for 14 days 2°C for Reduced chilling-related injury, Gonzalez-Aguilar et al.
5 days increased color development, higher (2001)
sugars, TSS, and ascorbic acid
contents
“Tommy Atkins” 10−4 M 5°C for 21 days + 5 days Reduced ion leakage and chilling- Gonzalez-Aguilar et al.
shelf related injury; increased shelf-life (2000)
and TSS
Nectarines — 1000 ppm 20°C for 4 days shelf Increased PAL activity, phenolics, Heredia and Cisneros-
and antioxidants Zevallos (2009)
Papaya “Khakdam” 0.39 mM PDJ 6 and 12°C for 4 days Reduced chilling-related injury, Kondo et al. (2005)
increased antioxidative enzymes
“Sunrise” 10−5 and 10−4 M 10°C for 32 days + 4 Inhibited fungal decay and reduced Gonzalez-Aguilar et al.
days shelf chilling-related injury (2003)
Advances in Postharvest Fruit and Vegetable Technology
Peach “Baifeng” 1 µM 0°C for 5 weeks Reduced chilling-related injury and Jin et al. (2009)
inhibited activities of PPO, POD;
increased activities of SOD, CAT,
and increased phenolics
“Jiubao” 0.1 mM 5°C for 21 days with Reduced chilling-related injury; Meng et al. (2009)
3 day shelf higher activity of POD and
increased SSC/TA ratio
“Stark Red Gold” 0.22 mM PDJ Preharvest application Reduced ethylene production, Ziosi et al. (2009)
softening, increased level of PA
Pear 1000 ppm 20°C for 4 days Increased PAL activity, phenolics, Heredia and Cisneros-
AOX, and antioxidants Zevallos (2009)
“La France” 0.39 mM PDJ 20°C for 9 days Increased ethylene production, Kondo et al. (2007)
Methyl Jasmonate in Postharvest

expression of ACS and ACO

Pineapple “Pattavia” 10−5 M 10°C for 21 days Reduced chilling-related injury, fruit Nilprapruck et al. (2008)
weight loss, and electrolyte leakage
“Red Spanish” 10−4 M 7°C for 12 days Decreased microbiological growth Martinez-Ferrer and
Harper (2005)
Plum “Amber Jewel”, 10−3 M During ripening for Increased SSC, respiration rates, Khan and Singh (2007)
“Angelino”, “Black 13 days ethylene production, activities of
Amber” ACS and ACO enzymes
“Black Amber” 1500 mL 0°C for 4 weeks Increased firmness and phenolics Ozturk et al. (2012)
“ Fortune” 1120 mg/L 0°C for 4 weeks Increased total phenolics, Karaman et al. (2013)
antioxidant, and chlorogenic acid
— 1000 ppm  20°C for 4 days Increased PAL activity, phenolics, Heredia and Cisneros-
and antioxidants Zevallos (2009)
Pomegranate “Malas Yazdi” 0.4 mM MJ, 1%–2% 0.5°C for 2 months Reduced chilling-related injury, Mirdehghan and Ghotbi
“Malas Ashkezar” CaCl2, 1–2 mM SA electrolyte leakage, and increased (2014)
TSS
(Continued)
223
 224

TABLE 10.1  (Continued)

Effect of Exogenous Application of Methyl Jasmonate on Postharvest Quality of Fruit
Fruit Cultivar MJ Concentration Conditions Inferences Reference
“Mollar de Elche” 0.01 and 0.1 mM 84 days Increased total phenolics and Sayyari et al. (2009)
anthocyanins; reduced chilling-
related injury and pitting
Raspberry “Jewel” “Autumn 0.1 mM MJ + 0.01– 4 weeks Higher SSC, total sugars, fructose, Wang and Zheng (2005)
(black and Bliss” 0.1 mM MJ (two sprays) glucose, sucrose and lower TA,
red) malic acid, citric acid, flavonoids,
and antioxidant
Rose apple “Kheaw” 0.39 mM PDJ 6 and 12°C for 4 days Reduced chilling-related injury and Kondo et al. (2005)
increased anti-oxidative enzymes
Strawberry “Camarosa” 50 µM Storage at 85% RH Increased respiration, ethylene Perez et al. (1997)
production, color development,
anthocyanin, and β-caroten
synthesis
164 µL 4°C for 7 days Increased antioxidants, reduced Flores et al. (2013)
nitrite production
“Pajaro” 50 µM — Increased ethylene production and Mukkun and Singh (2009)
ACO activity
— 1000 ppm 20°C for 4 days Increased PAL activity, phenolics, Heredia and Cisneros-
AOX, and antioxidants Zevallos (2009)
Sweet Cherry — 2 mM SA + 0.2 mM MJ Storage at 25°C Reduced disease, increased Yao and Tian (2005b)
β-1,3-glucanas, PAL, POD
activities
Advances in Postharvest Fruit and Vegetable Technology
Tomato “Ailsa Craig” 10 mM for 10 min 25°C for 14 days Inhibited gray mold, stimulated CAT, Zhu and Tian (2012)
APX gene expression, enhanced
ASC and GSH contents
“Beefsteak” 22.4 µM 0°C for 21 days Reduced chilling-related injury and Fung et al. (2006)
increased levels of AOX
“Beefstake” 0.01 mM 5°C for 4 weeks Alleviated chilling-related injury, Ding et al. (2001)
increased HSPs, especially class II
HSPs
“Castlemart” 0.5 µM 15 days Increased lycopene biosynthesis and Liu et al. (2012)
over-expression of lycopene
biosynthetic genes
Tomato — 10−4 m With MAP for 9 weeks Delayed ethylene production, Siripatrawan and
Methyl Jasmonate in Postharvest

reduced softening and chilling- Assatarakul (2009)

related injury
“Charleston” 220 µL/L 10°C for 27 days More color development, firmness, Baltazar et al. (2007)
reduced CO2 and ethylene
production

Note: DAFB  = days after full bloom, PPO = polyphenol oxidase, SOD = superoxide dismutase, POD = peroxidise, TPC = total phenolic content, CAT = catalase.
BTH = benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester, TSS = total soluble solids, TA = titratable acidity, AOX = alternative oxidase, HSPs = heat-shock
proteins, PDJ = n-propyl-dihydrojasmonate, PAL = phenylalanine ammonia lyase, GABA = gamma-aminobutyric acid, LOX = lysyl oxidase.
225
226 Advances in Postharvest Fruit and Vegetable Technology

“Tommy Atkins” mango increased SSC in ripe fruit over untreated fruit (Fan et al.
1998b; Gonzalez-Aguilar et  al. 2000, 2001; Herrera et  al. 2004). Similarly, post-
harvest MJ-treated Japanese plum fruit exhibited improved fruit quality, including
color, ascorbic acid, total carotenoids, and SSC (Khan and Singh 2007).
The effect of MJ application on the sugar content of fruit reported in the literature
is controversial. Higher glucose and fructose levels were reported in “Kent” mango
fruit treated with 10−4 M MJ (Gonzalez-Aguilar et  al. 2004) but in another study
on “Kent” mango, fruit treated with 10−4 M MJ increased the sucrose level without
influencing fructose and glucose levels, while 10−5 M MJ decreased fructose and
glucose without any change in sucrose (Gonzalez-Aguilar et al. 2001).

10.6.4 Phytochemicals
Phytochemicals in the fruit can be improved significantly by MJ treatment. Examples
of this action are accrual of resveratrol in grapes; and accumulation of anthocya-
nins and antioxidants, alkaloids, and saponins; and taxol formation in various fruits
(Ahuja et al. 2012; Verpoorte et al. 2000; Wang and Zheng 2005). MJ was found to
stimulate the activity of PAL in guava, which led to accumulation of total phenolic
compounds, while in tomato and guava fruit, MJ enhanced LOX activity (Gonzalez-
Aguilar et al. 2004; Heitz et al. 1997). Postharvest application of MJ vapor increased
levels of ascorbic acid, total carotenoids and total antioxidants, in Japanese plums
(Khan and Singh 2007).

10.6.5 Oxidative Stress
MJ treatment has been found to increase antioxidant enzyme activity of many sys-
tems, including ascorbate peroxidase, superoxide dismutase, glutathione peroxidase,
dehydro ascorbate reductase, guaiacol peroxidase, glutathione reductase, and mono-
dehydro ascorbate reductase (Chanjirakul et al. 2006). Treatment with MJ increased
the level of polyphenols of guava, and enhanced catechin, chlorogenic acid, epicate-
chin, and phloridzin levels in apple (Gonzalez-Aguilar et al. 2004; Rudell et al. 2002).
Ascorbic acid is an antioxidant, and MJ application enhanced its level in various
fruit (Gonzalez-Aguilar et al. 2004). Moreover, it has been shown that MJ treatment
enhanced the transcription of genes involved in ascorbic acid de novo synthesis in
plant-cell suspensions. Treatment with MJ vapor before cold storage also enhanced
expression of alternative oxidase genes that are related to reduction of ROS forma-
tion, which could affect the marked increased tolerance of fruit to chilling-related
injury (Fung et al. 2004).

10.7  MJ AND PHYSIOLOGICAL DISORDERS AND DISEASES

Limitation on the use of low-temperature storage is often due to the occurrence of
flesh-softening, internal breakdown, or fruit decay during storage or in poststorage
handling. Potential applications of MJ in the prevention of chilling-related injury and
other postharvest disorders and diseases have been documented in Table 10.1. Some
aspects are discussed below.
Methyl Jasmonate in Postharvest 227

10.7.1 Chilling-Related Injury
Use of MJ and JA to ameliorate the sensitivity of fruit to develop chilling-related
injury was first reported by Wang and Buta (1994), and has since been demonstrated
in many commodities. Some examples of this effect are MJ applied as a dip or vapor
to avocado and grapefruit has been reported to decrease chilling-related injury dur-
ing storage (Meir et al. 1996), while banana stored at 5°C for one week was protected
from development of chilling-related injury when treated with n-propyl-dihydrojas-
monate (PDJ) (Chaiprasart et al. 2002). MJ treatments also decreased decay in straw-
berry stored at 5°C and 10°C (Moline et al. 1997). Use of MJ in combination with
hot water is a common treatment protocol that not only reduces ion leakage but also
the incidence of chilling-related injury (Gonzalez-Aguilar et al. 2000, 2003, 2004).
However, MJ also increased abscission in the harvested cherry tomatoes, which is
considered undesirable, as cherry tomatoes are marketed attached to the peduncles.
Meir et al. (1996) proposed that MJ might interact with signal transduction cas-
cades of the chemical changes involved in the tolerance of chilling injury. It is now
known that MJ produces specific signals and activates certain enzymatic reactions
involved in the synthesis of proteins known to have significant roles in the posthar-
vest life of horticultural crops (Buta and Moline 1998; Droby et al. 1999; Gonzalez-
Aguilar et al. 2004).
A role for ethylene is implicated in a study of bananas treated with PDJ before
storage at 5°C reduced chilling-related injury after cold storage and at 20°C for one
week during ripening. Compared to control, treated bananas exhibited increased
ethylene evolution and it can be hypothesized that this might play an important role
in inhibiting chilling-related injury, although more evidence is required to support
this hypothesis (Ding et al. 2002). Alternately, MJ also induces mRNA accumulation
of heat-shock proteins (HSPs) in tomato that can partly explain reduction in chilling-
related injury symptoms (Ding et al. 2001). Exposure of the heated fruit to ambient
temperature conditions prior to transfer under the low temperatures eliminates the
tolerance capability to low temperature or chilling-stress (Sabehat et al. 1996). Thus,
biochemical and physiological changes instigated by MJ treatment might vary when
the target produce is stored at cold or ambient temperature after treatment.

10.7.2 Inhibition to Disease
Fungal diseases can be controlled by synthetic fungicides, but development of resis-
tance by the pathogens and escalating consumer concerns about chemical use and
residues have stimulated the search for alternative measures to control fruit diseases
(Fan et al. 1998b). One such approach is to utilize methods that stimulate the natural
defense systems of plants to invading fungi (Meir et al. 1998). JAs are involved in
various signal transduction systems and result in the induction of defense-responses
against pathogen attack (Gundlach et al. 1992; Guo et al. 2014; Nojiri et al. 1992; Yao
and Tian 2005b). Application of JA has been reported to induce resistance against a
range of organisms in a range of fruit. Some examples are: resistance to B. ­cinerea,
C.  acutatum, and A. brassicicola, in papaya, loquat, and tomato, respectively
(Cao et al. 2008; Gonzalez-Aguilar et al. 2003), while application of MJ increased
228 Advances in Postharvest Fruit and Vegetable Technology

resistance against A. alternata and B. cinerea in strawberry (Moline et al. 1997) and
eliminated decay in raspberry fruit (Wang 2003).
MJ treatment in combination with C. laurentii also exhibited positive effects for
the control of blue mold and brown rot of peaches (Yao and Tian 2005a). This inhibi-
tory mechanism is considered to be due to C. laurentii and to MJ-induced resistance.
Yao and Tian (2005a) reported that direct inhibition of blue mold growth in vitro
was due to the application of MJ. A probable mode of action of MJ in the induc-
tion of disease resistance might be due to phenol and anthocyanin synthesis and
the antagonistic effects of ethylene (Gonzalez-Aguilar et al. 2004; Meir et al. 1998;
Rudell et al. 2002).

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 11 Postharvest Oxidative
Stress in Fresh Fruits
Sukhvinder Pal Singh

CONTENTS
11.1 Introduction................................................................................................... 237
11.2 Mechanisms for ROS Scavenging................................................................. 238
11.2.1 Enzymatic Antioxidants.................................................................... 239
11.2.2 Nonenzymatic Antioxidants.............................................................. 239
11.3 Postharvest Oxidative Stress......................................................................... 241
11.4 Factors Affecting Postharvest Oxidative Stress............................................ 243
11.4.1 Genotype/Cultivar............................................................................. 243
11.4.2 Harvest Maturity................................................................................244
11.4.3 Storage Temperature and Duration....................................................246
11.4.4 Storage Atmospheres.........................................................................248
11.4.5 Postharvest Treatments...................................................................... 252
11.4.5.1 1-Methylcyclopropene......................................................... 252
11.4.5.2 Nitric Oxide........................................................................ 253
11.5 Conclusions.................................................................................................... 254
References............................................................................................................... 255

11.1 INTRODUCTION
Oxygen (O2) is the most abundant molecule in the biological system and is often a
source of free radicals as its partially reduced species are generated through normal
metabolic processes. Most of the O2 consumed by nonphotosynthetic plant tissues
is reduced to water by the terminal oxidase(s) of the respiratory electron transport
chain in the mitochondria (Apel and Hirt, 2004). Reactive oxygen species (ROS)
are the partially reduced forms of molecular oxygen that result from either the exci-
tation of O2 to form singlet oxygen or the transfer of one, two, or three electrons to
O2 to form, respectively, superoxide (O2− ), hydrogen peroxide (H2O2) or hydroxyl
radical (OH·) (Hodges et al., 2004). Both O2− and OH· are extremely reactive and
can cause oxidative injury leading to cell death. The average life span of these ROS
varies from nanoseconds (e.g., OH) to milliseconds (e.g., O2− , H2O2). The OH· can
also be generated by the interaction of O2− and H 2O2 in the presence of transition
metal ions, so called “Haber-Weiss reaction.” Cells do not possess detoxification
mechanism for OH· due to its very high reactivity, and rely on mechanisms prevent-
ing its formation. These mechanisms include the preceding elimination of O2− and

237
238 Advances in Postharvest Fruit and Vegetable Technology

H2O2 and/or sequestering metal ions that catalyze the Haber-Weiss reaction with
metal-binding proteins such as ferritins or metallothioneins (Gechev et al., 2006).
In addition to H2O2, O2− can also react with nitric oxide radical (NO·) to form per-
oxynitrite (ONOO−), which can rapidly protonate to peroxynitrous acid (ONOOH),
a powerful oxidizing agent. The reactions among various types of ROS can, there-
fore, generate intermediates or products that are capable of causing extreme levels
of oxidative injury to the cell.
There are multiple sites and sources of ROS production in a plant cell, adding to
the complexity of complete understanding of their chemistry and metabolism. As a
part of normal metabolism, ROS levels are maintained at basal levels, except during
stress conditions and developmental signals. The major areas of aerobic biochemis-
try (e.g., respiratory and photosynthetic electron transport; oxidation of glycolate,
xanthine, and glucose) are involved in ROS production, in addition to their gen-
eration by several enzyme systems (e.g., plasmalemma-bound NADPH-dependent
superoxide synthase and SOD) (Noctor and Foyer, 1998). Chloroplasts are the major
sites of ROS production in photosynthetic tissues. The over-energization of photo-
synthetic electron transfer chains cause production of O2− , mainly by electron leakage
from Fe–S centers of photosystem-I or reduced ferrodoxin to O2 (Mehler reaction)
(Apel and Hirt, 2004; Gechev et al., 2006). The O2− is rapidly metabolized into H2O2
by the action of SOD. Under excess light conditions, the photoinhibition of photosys-
tem-II also causes the drastic increase in singlet oxygen production. The importance
of chloroplastic sources may, however, decrease in ripening fruits and commodities
placed in dark storage (Hodges et al., 2004). Mitochondria are the major consum-
ers of O2 and, therefore, contribute to the production of ROS (Møller, 2001; Navrot
et al., 2007). During stress conditions, high O2 concentration may build up in the cell
due to reduced respiratory activities. Consequently, the high redox status of the elec-
tron transport chain (ETC), and high intra-mitochondrial O2 concentration favor the
leakage of electrons from single-electron-reduced components to molecular oxygen,
resulting in the increased production of ROS. The potential sites of the leakage of
single electrons to molecular O2 are complex I and complex III (Navrot et al., 2007).
Peroxisomes and glyoxysomes also contribute to the ROS generation in the cell dur-
ing photorespiration and fatty-acid oxidation, respectively.

11.2  MECHANISMS FOR ROS SCAVENGING

Cellular homeostasis is achieved by a delicate balance among different pathways
operating in various organelles. In a plant cell, ROS are produced during normal
metabolism as well as stress conditions. The concentration of ROS in the cell is an
important factor in determining their role as beneficial molecules in various signal
transduction processes or in causing oxidative damage (Suzuki and Mittler, 2006).
ROS has the ability to initiate cascade reactions and their products and intermedi-
ates result in damage to the lipids, proteins, and DNA. Therefore, the level of ROS is
regulated by scavenging them to avoid accumulation to toxic levels in the cell. The
scavenging mechanism of these ROS is also as complex and diverse as their genera-
tion sites. It involves both enzymatic and nonenzymatic antioxidant systems to act
cooperatively to mitigate the ROS levels (Hodges et al., 2004).
Postharvest Oxidative Stress in Fresh Fruits 239

11.2.1 Enzymatic Antioxidants
The enzymatic antioxidants include various enzymes such as superoxide dismutase
(SOD), catalase (CAT), guaiacol-peroxidase (POD), ascorbate peroxidase (APX),
monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR),
glutathione reductase (GR), glutathione peroxidase (GPX), and glutathione-s-trans-
ferase (GT), which are responsible for direct or indirect scavenging of ROS (Noctor
and Foyer, 1998). SOD, CAT, and POD are known as primary antioxidant enzymes,
which serve as frontline defense antioxidants. The dismutation of O2− into H2O2 is
catalyzed by SOD. The activity of SOD converts a ROS into another type of ROS,
H2O2, which is subsequently detoxified into water (H2O) and O2 by the CAT and APX.
CAT is considered to be more efficient as it does not require any reducing power for
H2O2 detoxification as opposed to guaiacol-POD and APX, but it has low substrate
affinity (Apel and Hirt, 2004). The activity of APX requires an ascorbate (AA) and
glutathione (GSH) regeneration system, which is also called ascorbate-glutathione
cycle. APX catalyzes the conversion of H2O2 into H2O using two molecules of AA as
a reducing power with a concomitant production of two molecules of monodehydro-
ascorbate (MDHA). MDHA radicals rapidly disproportionate into dehydroascorbate
(DHA) and AA; the latter reaction is catalyzed by MDHAR using NADPH as the
electron donor. The DHA is reduced back to AA by the action of DHAR, using GSH
as the reducing agent. Finally, GR can regenerate GSH from glutathione disulfide
(GSSG) using NAD(P)H as a reducing agent. The efficient removal of ROS by the
enzymatic antioxidants is therefore dependent on low molecular weight reductants,
AA and GSH, because these supply electrons necessary for the activity of enzymes
(Noctor and Foyer, 1998).
In addition to other enzymatic antioxidants, plant mitochondria have evolved
another system of mitigating the production of ROS during stress conditions (Purvis,
2004). In addition to cytochrome oxidase, plant mitochondria have an alternate oxi-
dase (AOX), which is located on the matrix side of the inner membrane, and oxidizes
ubiquinol directly, without producing a proton electrochemical gradient across the
inner membrane. Therefore, electron flow through the AOX is not coupled to the
synthesis of ATP and causes the reduction in redox potential of the component of
ETC and lowers the concentration of O2 in the mitochondria. This overall process
reduces the potential for leakage of electrons from the system, especially under the
enhanced respiration experienced both during and after stress (Purvis, 2004). The
up-regulation of AOX activity in response to various environmental stimuli can thus
contribute to the reduction in ROS production in the cell.

11.2.2 Nonenzymatic Antioxidants
Ascorbate (AA) is one of the most abundant low-molecular-weight antioxidants pres-
ent in plant tissues (Davey and Keulemans, 2004). AA can donate electrons, which
makes it an effective ROS-detoxifying compound in a wide range of enzymatic and
­nonenzymatic reactions in the aqueous phase. AA serves as an antioxidant to scavenge
ROS which oxidizes AA to MDHA and DHA. MDHA and DHA can be converted
back into AA to maintain the cellular pool by the action of MDHAR and DHAR,
240 Advances in Postharvest Fruit and Vegetable Technology

respectively. Therefore, the amount of AA present in the cell is modulated by both

its biosynthesis and oxidation loss. The ratio of AA to DHA is an indicator of the
redox potential of the cell and can also serve as a marker for the degree of oxida-
tive stress (Davey and Keulemans, 2004). AA also regenerates tocopherols from the
tocopheroxyl radical thereby providing membrane protection (Blokhina et al., 2003). It
also plays important roles in resistance to a number of environmental stresses, such as
pathogen infection, hypoxia stress, high light and UV-B radiation (Noctor and Foyer,
1998). It has been implicated in the regulatory role in some fundamental cellular pro-
cesses such as photo-protection, cell-cycle, and cell expansion (Blokhina et al., 2003).
Glutathione (GSH) is a nonprotein sulfur-containing tripeptide (Glu-Cys-Gly)
and acts as a storage and transport form of reduced sulfur (Tausz et al., 2004). GSH
is related to the sequestration of xenobiotics and heavy metals, and is also an essen-
tial component of the cellular antioxidative defense system, which keeps ROS under
control. Being an integral component of the AA-GSH cycle, it is involved in regen-
eration of AA during the removal of excess H2O2 from the cell. Its role in regenera-
tion of another antioxidant, AA, provides it a central role in the antioxidant defense
system. In addition to AA-GSH cycle, GSH is possibly involved in degradation of
H2O2 in a reaction catalyzed by GPX, but the role of GSH as a substrate of GPX
is still questionable (Szalai et  al., 2009). GSH plays a very important role in the
removal of lipid peroxides through the activity of glutathione-s-transferases (GT).
The accumulation of GSSG, the oxidized form of GSH, leads to a decrease in GSH/
GSSG ratio, which symbolizes the oxidized environment and the reduction of anti-
oxidant capacity of the glutathione system (Noctor and Foyer, 1998).
Polyphenolic compounds, a group of plant secondary metabolites of >9000
individual molecules, are involved in an array of processes, including plant–patho-
gen interactions, pollination, light-screening, seed development, and allellopathy
(Hernández et al., 2009). Many biosynthetic genes for polyphenolic compounds are
induced under stress conditions and, accordingly, their levels increase during expo-
sure to biotic and abiotic stresses, such as wounding, drought, metal toxicity, and
nutrient deprivation. In vitro antioxidant tests reveal that the antioxidant capacities
of phenolic compounds are several-fold higher than those of ascorbate (vitamin C)
or α-tocopherol (vitamin E), two well known in planta antioxidants. Phenolic com-
pounds have been suggested to act as antioxidants, protecting plants from oxidative
stress. Phenolic compounds and peroxidases are generally localized in vacuoles of
plant cells. When H2O2 is formed in vacuoles or tonoplasts, or when H2O2 formed
outside of vacuoles is diffused into the organelles, vacuolar peroxidases oxidize phe-
nolics, especially flavonols and hydroxycinnamic acids, to form phenoxyl radicals
(Takahama, 2004). Phenoxyl radicals formed by peroxidase-dependent reactions are
reduced by AA in vacuoles. This suggests that ascorbate/phenolics/peroxidase sys-
tems in the vacuoles can scavenge H2O2.
Tocopherols are a group of closely related phenolics benzochroman derivatives
having extensive ring alkylation (Lurie, 2003). There are four tocopherols isomers
(α-, β-, γ-, and δ-); α-tocopherol has the highest antioxidant activity among all. The
molecule is amphipathic in nature; the hydrophobic tail is located in a membrane
and is associated with the acyl chains of fatty acids or their residues, while the
polar chromanol head group lies at the membrane–cytosol interface. It acts as an
Postharvest Oxidative Stress in Fresh Fruits 241

antioxidant either by chemical scavenging of free-radicals or by physical deactiva-

tion, thereby preventing the proliferation of oxidative chain reactions. The major role
of α-tocopherol is to protect the biological membrane by acting as a chain-reaction
terminator for the removal of polyunsaturated fatty acid (PUFA) radicals generated
during lipid peroxidation (Blokhina et al., 2003).
Carotenoids are among the most common natural pigments in plants and play a
major role in the protection against photooxidative processes (Lurie, 2003). These
are very efficient antioxidants scavenging singlet molecular oxygen and peroxyl radi-
cals and, thus, prevent oxidative damage to the tissue. In plant tissues, carotenoids
mostly occur in the chloroplast membranes and help prevent oxidative injury from
ROS produced in the photosynthetic electron transport chain. The richness of chlo-
roplast membranes with PUFA linolenate makes them more prone to peroxidation.
Therefore, the presence of carotenoids prevents the membrane damage to the chlo-
roplast by ROS (Lurie, 2003).

11.3  POSTHARVEST OXIDATIVE STRESS

Oxidative stress occurs when critical balance is disrupted by excess of ROS, reduc-
tion in antioxidants, or both. The important homeostatic parameters must be set to
counteract the oxidant effects and to restore redox balance by the cell. There are sev-
eral factors that augment the development of oxidative stress in plants and include,
but are not limited to, temperature extremes, drought and flooding, salinity, ozone
exposure, UV irradiation, heavy metal toxicity, herbicides, and environmental pol-
lutants (Apel and Hirt, 2004). These environmental perturbations enhance the ROS
production in the plant or plant organs. If ROS production exceeds the antioxidant
capacity of the tissue, it causes oxidative damage to the cell, which ultimately leads
to cell death.
Fresh fruits are living entities that continue to respire and transpire even after
harvest. The postharvest physiological and biochemical changes have both desirable
and undesirable effects on fruit quality. The most important desirable changes in
climacteric fruit are brought by the ripening process, which involves the changes in
skin and flesh color, texture, taste, and emission of aroma-volatile compounds. The
advanced stages of ripening culminate into the senescence process, which eventually
leads to the death of fruit, and is the most undesirable from economic viewpoint.
On the other hand, nonclimacteric fruit are generally harvested at the ripe stage, but
there are still some desirable postharvest changes in the development of fruit texture
and flavor. Similar to climacteric fruits, the onset of senescence in nonclimacteric
fruits is also a natural process that leads to death. Fruit-ripening has been described
as an oxidative phenomenon that involves production and removal of ROS from the
tissue, and the failure or reduction in the capability of the antioxidant system leads
to the oxidative injury coinciding with the onset/advancement of senescence (Masia,
1998; Singh et al., 2012).
The objective of a postharvest technology is to slow down the ripening and/or
delay the onset of senescence in fruit and to deliver a high-quality fruit to the con-
sumer/processor. The postharvest life of a fruit can be manipulated by regulating
its ripening and senescence rates. The better understanding of the physiology of
242 Advances in Postharvest Fruit and Vegetable Technology

ripening and senescence of fruit can, therefore, lead to the development of more
effective postharvest practices aimed to minimize qualitative and quantitative
losses. Low temperatures have been widely used as a means to retard the biological
activity of fruit that enables its storage, transport and distribution over longer periods
and distances. Studies have shown that chilling temperatures can potentially cause
oxidative injury to the fruit, depending on the storage temperature and length of
exposure. Most of the postharvest practices aimed to extend the storage or shelf-life
of produce have been known to act as stress factors. It is therefore obvious that the
development of oxidative stress in postharvest situations is indicative of the signifi-
cant stress experienced by a commodity (Toivonen, 2004).
Some of the postharvest physiological and biochemical changes during the supply
chain result in development of certain disorders that affect the consumer accept-
ability of fruit. The commercially important storage disorders, such as superficial
scald in apple, flesh and core browning in apples and pears, and internal browning
in plums, have been attributed to the adverse effects of postharvest oxidative stress
in fruit. Chilling-related injury is also one of such major physiological disorders
that limit the long-term cold-storage of fruits below a critical threshold temperature
and adversely affect the consumer experiences, especially when the symptoms are
invisible or internal. Fruits of tropical and sub-tropical origin are generally more
susceptible to chilling-related injury than their temperate/Mediterranean counter-
parts. The development of chilling-related injury in temperate/Mediterranean fruits
is generally a function of duration of exposure to a particular temperature. There
is increasing evidence that chilling-related injury in fruits is also a result of oxida-
tive damage to the membrane, which results in irregularities in the physiology and
metabolism of the cell.
A large volume of literature has accumulated on the importance of antioxidant
metabolism in relation to fruit quality and durability in postharvest supply chain.
The sites of ROS production and their roles in cell physiology are complex and intri-
cate in nature, and so are the antioxidant defense systems evolved by the plant or
plant organs in order to cope with the environmental stresses. Postharvest oxidative
stress in fruits can be detected directly and indirectly (Toivonen, 2004). The degree
of oxidative stress can be directly determined by the measurements of accumulation
of ROS, increases in lipid peroxidation products, enhanced membrane disintegra-
tion or accumulation of brown pigments. The indirect measurements include the
determination of dynamics of nonenzymatic and enzymatic antioxidant systems.
The understanding of inter-relationship of oxidative stress parameters and their
association with storage disorders offer attractive alternatives to develop the inno-
vative postharvest practices. The comprehensive measurement of various compo-
nents of antioxidant protection systems is, therefore, central to demonstrating their
role in fruit quality with regard to physiological disorders. As a general rule, plants
have two primary strategies of coping with oxidative stress: avoidance and tolerance
(Hodges et  al., 2004). Postharvest fruits are themselves not in situations to adopt
the ­“avoidance” strategy, but the human interventions to mitigate the conditions
promoting ROS production can contribute to “avoidance.” However, “tolerance” to
oxidative stress of postharvest fruits has been associated with their inherent antioxi-
dant potential. But there are several other factors that affect the capability of fruits
Postharvest Oxidative Stress in Fresh Fruits 243

to encounter postharvest oxidative stress and some of these important factors are
reviewed and discussed in the following sections.

11.4  FACTORS AFFECTING POSTHARVEST OXIDATIVE STRESS

11.4.1 Genotype/Cultivar
Postharvest behavior of a cultivar is determined by its genetic makeup. Some culti-
vars with a faster rate of ripening and senescence have poor postharvest storage and
shelf-life. Generally, the cultivars with higher antioxidant potential have better stress
resistance, nutritional quality, and storage characteristics (Davey and Keulemans,
2004; Łata et al., 2005). Screening of apple cultivars for some enzymatic and non-
enzymatic antioxidants revealed a great variation in the activities and concentration
of these antioxidants among cultivars at harvest and following cold storage (Davey
et al., 2004; Davey and Keulemans, 2004; Łata et al., 2005). As compared to vari-
ation at harvest, apple cultivars differed substantially in their ability to maintain
L-AA levels during storage. Generally, cultivars that could maintain their AA and
GSH pools also had better storage properties (Davey and Keulemans, 2004). For
instance, “Sunrise” and “Gravenstein” cultivars showed maximal losses of AA and
GSH—by about 80% and 50%—following shelf-life and these cultivars are known
to have poor storage qualities, with susceptibility to browning and rot. For cultivars
such as “Arlet” and “Angold,” there was no significant difference between AA at
harvest, cold storage or shelf-life, and could be stored for up to six months at 1°C.
Some cultivars such as “Greenstar” and “Braeburn” were able to maintain or actu-
ally slightly increase both AA and GSH levels under both cold storage and shelf-life
and retain quality for up to six months at 1°C (Davey and Keulemans, 2004). Other
researchers have also shown a great variation in the concentration of antioxidants in
different cultivars of apples (Łata et al., 2005).
The concentrations of antioxidants also differ with the fruit tissue. AA and GSH
levels in the peel tissue were 6.7- and 2.8-fold higher, respectively, than those in
the cortical tissue (Davey et al., 2004). Similarly, in 25 cultivars of apples, on aver-
age, GR, CAT, AA, GSH, and phenolics were approximately 2.9, 1.5, 4.4, 1.7, 2.1,
2.1, and 2.5 times higher in the apple peel than the whole apple fruit (Łata et al.,
2005). A recent study also confirmed that three apple cultivars, “Delicious,” “Golden
Delicious,” and “Gala” showed higher AA content in peel than in cortical tissue as
the peel tissue has to play a protective role in response to both abiotic and biotic
stresses such as ultraviolet radiation, wind, pathogens, and insects (Felicetti and
Mattheis, 2010). Similarly, Japanese plum cultivars have been reported to show vari-
ations in their oxidative and antioxidative systems during fruit ripening. Climacteric-
type cultivars, “Blackamber” and “Amber Jewel,” showed a faster decline in the
ability of enzymatic and nonenzymatic antioxidative systems, parallel to the faster
rate of ripening and senescence, as compared to the suppressed-climacteric cultivar,
“Angeleno” (Singh et al., 2012).
Cultivar is also a major factor in determining the susceptibility of fruit to post-
harvest physiological disorders. For instance, apple cultivars greatly differ in their
susceptibility to superficial scald, a very serious postharvest physiological disorder.
244 Advances in Postharvest Fruit and Vegetable Technology

Cultivars such as “Empire,” “Gala,” “Golden Delicious,” and “Crispin” are resistant
to scald development, whereas others such as “Delicious,” “McIntosh,” “Cortland,”
“Granny Smith,” “Idared,” “Rome Beauty,” and “Fuji” are susceptible to this disor-
der (Emongor et al., 1994). It is believed that the biosynthesis of α-farnesene and its
subsequent degradation to conjugated trienes (CT) may cause the disruption, discol-
oration and death of surface cells (MacLean et al., 2006). Scald development may
also be related to ROS production either through low-temperature stress or through
α-farnesene metabolism. No clear relationships between antioxidant enzyme activi-
ties and susceptibility to superficial scald development have been observed in differ-
ent apple cultivars (Du and Bramlage, 1994, 1995; Meir and Bramlage, 1988). The
confounding effects of cultivar, maturity, ripeness, and environmental conditions,
may be ascribed to obstruct the development of a clear relationship between the scald
development and antioxidant systems in peel (Rao et al., 1998). Some studies have
shown that activities of the H2O2-degrading enzymes, POD and APX, were higher in
scald-resistant selections of “White Angel” × and “Rome Beauty” apples, indicating
the role of ROS in induction of scald (Rao et al., 1998). The amount of α-farnesene
produced during scald development may be less important than the activity of the
antioxidant enzyme system (Whitaker et al., 2000). To reduce the incidence of super-
ficial scald in apples, the success of postharvest treatments of fruit with antioxidants
and storage under low O2 atmospheres support the notion that antioxidant metabo-
lites and/or enzymes may influence the resistance to scald (DeLong and Prange,
2003). The studies indicate that scald is characterized by cellular oxidation within
the hypodermis, and oxidative stress theory can partially explain the susceptibility
to this disorder exhibited by different cultivars of apples.
“Clemenules” and “Clementine” cultivars of mandarins are considered to be
chilling-tolerant compared to “Nova” and “Fortune” cultivars that are chilling-
sensitive (Sala, 1998). The tolerance to chilling in these cultivars has been attrib-
uted to the increased activities of CAT, APX, and GR in response to chilling-related
stress. Further studies on the role of antioxidant defense showed that CAT is the
major antioxidant enzyme involved in the defense mechanism of mandarin fruits
against chilling-related stress (Sala and Lafuente, 2000). In another study on pepper
(Capsicum annuum L.) fruit, susceptibility to chilling-related injury appeared to be
related to the activities of SOD and CAT, which were much higher in “Buchon,” a
chilling-tolerant cultivar, than “Nockgwang,” a chilling-sensitive cultivar (Lim et al.,
2009). These studies suggest that cultivars with tolerance to chilling-related stress
are generally equipped with a more efficient antioxidative system.

11.4.2 Harvest Maturity
Harvest maturity has been shown to affect the development of postharvest oxida-
tive stress in fruits, thereby influencing the storage potential and susceptibility to
oxidative injury (Toivonen, 2003a). The development of postharvest physiological
disorders in fruit crops has been related to the antioxidant levels at harvest and the
changes in their concentrations during cold storage. The accumulation of lipid-sol-
uble antioxidants in apple peel, due to delayed harvest, has been shown to decrease
the incidence of superficial scald during cold storage (Barden and Bramlage, 1994;
Postharvest Oxidative Stress in Fresh Fruits 245

Diamantidis et  al., 2002), but a clear relationship between antioxidant enzymes
and scald-resistance has not been established, due to contradictory reports (Du and
Bramlage, 1995; Rao et al., 1998). However, the flesh-related disorders in apples and
pears have been shown to increase with advanced maturity. The decrease in activi-
ties of SOD and CAT in flesh tissue increased the incidence of Braeburn browning
disorder (BBD) in late-harvested “Braeburn” apples (Toivonen et al., 2003). Another
study showed that the changes in activities of antioxidant enzymes, SOD, CAT, and
POD, in the flesh tissue of “Golden Smoothee” apples were mainly influenced by
the climatic conditions during the last phase of on-tree fruit maturation (Molina-
Delgado et al., 2009). The cooler season resulted in a higher antioxidant potential of
the fruit in terms of higher activities of these enzymes and AA content. In the cooler
season, the delay in harvesting caused a significant decrease in the activities of SOD
and POD, and AA levels in the flesh tissue. In general, the changes in quality param-
eters during on-tree ripening were not related to the capability of the fruit to produce
ethylene, but rather to endogenous levels of antioxidants, especially CAT and AA, at
the earliest picking date (Molina-Delgado et al., 2009).
The decline in AA concentration in the flesh tissue of apple cultivars during the
final stages of fruit maturation has been demonstrated by various researchers (Davey
et al., 2007; Felicetti and Mattheis, 2010; Molina-Delgado et al., 2009). AA concen-
trations were also found to be negatively correlated with mean preharvest daytime
temperature; however, since preharvest temperature and harvest date themselves are
closely linked, it was not possible to definitively separate the relative contributions
of genetic and environmental factors to these traits (Davey et al., 2007). The delayed
harvesting of “Conference” and “Passa Crassana” pears increased susceptibility to
core browning due to a decrease, with advanced maturity, in the ability of the anti-
oxidant system to protect from ROS (Lentheric et al., 1999; Veltman et al., 1999).
The behavior of peel tissue and flesh tissue seems to be quite different with regards
to maturity and subsequent changes in respective antioxidant systems (Toivonen,
2003a).
It is well-known that sensitivity to chilling-related injury is strongly influenced
by the fruit’s maturity. Fruit at advanced stages of maturity or ripening are chill-
ing-tolerant compared to less mature or unripe fruit. In mangoes, the activities of
SOD, CAT, APX, and PPO in pre-yellow and yellow fruit were reported to be higher
than those of the green, from day six to day 12, during cold storage (Zhao et al.,
2009). A lower content of MDA, higher levels of AA and GSH were maintained
in pre-yellow and yellow fruit than that in green fruit. These results suggested that
chilling-tolerance of pre-yellow and yellow mangoes compared to green fruit was
due to their higher antioxidant potential (Zhao et al., 2009) and, therefore, cold stor-
age of mangoes at advanced stages of maturity can help alleviating chilling-related
injury to some extent. Another typical example is pepper (Capsicum annuum L.)
fruit. Pepper fruit harvested at mature green and breaker stages were more prone to
chilling-related injury than those at red-ripe stage (Lim et al., 2009; Lin et al., 1993).
The higher APX and SOD activities in mitochondria from ripe-red pepper fruit
might play a role in avoiding the accumulation of ROS generated in the mitochondria
(Jiménez et al., 2002). The higher activities of SOD and CAT have been correlated
with chilling-tolerance at the red-ripe stage in peppers (Lim et al., 2009). Cherries
246 Advances in Postharvest Fruit and Vegetable Technology

harvested at advanced stages of maturity, four days later than commercial harvest
dates, showed higher levels of phenolics and total antioxidant activity at harvest and
also after 16 days of cold storage at 2°C plus two days of shelf-life (Serrano et al.,
2009). In “Amber Jewel” plums, the incidence and severity of chilling-related injury
was higher in fruit from the delayed harvest compared to commercial harvest (Singh
and Singh, 2013c). The status of enzymatic and nonenzymatic antioxidative system
during cold storage of Japanese plums was reported to be more important in provid-
ing protection against oxidative injury (expressed as chilling-related injury) than the
at-harvest antioxidant status. Delayed harvested fruit experienced more oxidative
stress during cold storage compared to the fruit harvested at commercial maturity.
It has been widely argued that the changes in antioxidant components during cold
storage of fruit should be considered more important in providing protection against
oxidative injury than their at-harvest antioxidant status.

11.4.3 Storage Temperature and Duration

Storage temperature and duration are important factors that govern the develop-
ment of postharvest oxidative stress in fruits (Hodges et al., 2004; Toivonen, 2004).
Despite the inhibitory effects of certain postharvest procedures on fruit-ripening and
senescence, the storage potential of each fruit cultivar is definite. Storage at opti-
mum temperature is essential to maintain fruit quality. However, storage at tempera-
tures below and above the optimal limit accelerates the loss of fruit quality, either
by inducing chilling-related injury or by a faster rate of ripening and senescence.
Even storage life under optimum conditions is also limited to a certain time period,
depending on the cultivar and several other factors such as fruit maturity.
Antioxidant metabolism has been shown to play a very important role in determin-
ing the storage potential of fruit. The accumulation of ROS continues during posthar-
vest phase of fruit to a variable extent, depending upon the environmental conditions
and the antioxidant potential of the fruit. The production and accumulation of ROS
beyond the antioxidant capability of fruit tissue causes the oxidative damage, result-
ing in senescence and the appearance of visible injuries on the fruit. The impact of
storage-duration on the development of oxidative stress has been widely studied in
fruits, but there is relatively less information about the role of storage-temperature.
With the increase in storage-duration, there is an increased accumulation of ROS in
the fruit tissue. The increase in concentration of H2O2 has been reported to occur
during 16 weeks of cold storage at 0.5°C in different apple selections derived from
a cross between “White Angel” and “Rome Beauty” (Rao et al., 1998). The higher
levels of H2O2 have been associated with oxidative damage and the development of
flesh-browning in “Pink Lady”™ apples (De Castro et al., 2008), and also related
to water core in “Fuji” apples (Kasai and Arakawa, 2010). On the other hand, only a
transient increase in the concentration of H2O2 was observed in “Golden Smoothee”
apples during the first three days of storage, and then it declined continuously during
the subsequent 90 days of storage period (Vilaplana et al., 2006). Contrary to H2O2,
the accumulation of lipid peroxidation products continued during the entire storage-
duration, which indicated the development of oxidative stress with the progression
of storage (Vilaplana et al., 2006). The cultivar differences do exist in the storage
Postharvest Oxidative Stress in Fresh Fruits 247

behavior of fruits, especially with regard to accumulation of ROS, depending on

tissue type. The increase in ROS concentration and accumulation of lipid peroxida-
tion products as a function of storage duration have been reported in loquats (Cao
et al., 2009), peaches (Zheng et al., 2007), oranges (Huang et al., 2008), mangoes
(Singh and Dwivedi, 2008; Wang et al., 2009), and plums (Singh and Singh, 2012a,b,
2013a,b,c).
The activities of primary antioxidant enzymes, SOD, CAT, and POD, have been
known to change with respect to storage-duration. The activities of CAT and POD
increased significantly in the skin tissues of apple cultivars, “Empire,” “Cortland,”
and “Delicious” during the cold storage at 0°C for 12 or 24 weeks, while no sig-
nificant change in SOD activity was reported (Du and Bramlage, 1995). Similarly,
in flesh tissue, CAT activity has been reported to increase during cold storage of
“Golden Delicious,” “Golden Smoothee” and “Fuji” cultivars, with less impact on
SOD activity (Masia, 1998; Vilaplana et  al., 2006). CAT activity increased only
gradually in “Golden Delicious” while peaking in “Fuji” after 49 days of cold stor-
age. The decrease in CAT after 84 days of storage was coincident with the appear-
ance of water-core disorder in “Fuji” apples (Masia, 1998). Another study has shown
the decreases in activities of CAT and POD and increase in SOD activity during
16 weeks of cold storage of apple selections derived from a cross between “White
Angel” and “Rome Beauty” (Rao et al., 1998). These studies show mixed results in
terms of changes in the activities of various enzymes in even the same fruit tissue;
some were contradictory and others were in agreement with one another.
The changes in activities of various antioxidant enzymes have been studied in
other fruits such as mandarins (Sala, 1998; Sala and Lafuente, 2000, 2004), mangoes
(Wang et al., 2006, 2009), oranges (Huang et al., 2008), pears (Larrigaudière et al.,
2001b, 2004), peaches (Wang et al., 2004, 2005, 2006), plums (Singh et al., 2012;
Singh and Singh, 2013a), and raspberries (Chanjirakul et al., 2006). These studies
indicate that the increased activities of SOD, CAT, and POD enzymes are important
to regulate the ROS levels in the fruit tissue. The decreases in the activities of these
enzymes were generally coincident with the failure of the overall antioxidant system,
resulting in loss of membrane integrity, appearance of physiological disorders, and
termination of storage life.
AA concentrations generally decrease during cold storage of fruits. The decrease
in concentration of AA in apples has been reported to be a function of storage-dura-
tion (Barden and Bramlage, 1994; Davey et al., 2004, 2007; Davey and Keulemans,
2004; De Castro et  al., 2008; Fawbush et  al., 2009; Felicetti and Mattheis, 2010;
Łata et al., 2005) and a subsequent increase in the number of physiological disorders
(Davey et al., 2004; Davey and Keulemans, 2004; De Castro et al., 2008; Kasai and
Arakawa, 2010). The role of AA in better postharvest storage performance of pears
has been widely studied and accepted (Franck et al., 2003; Lentheric et al., 1999;
Veltman et  al., 1999, 2000). The factors that contribute to the core breakdown in
pears have been closely related to the loss of AA in the fruit (Franck et al., 2003).
In general, a significant decrease in GSH levels has been reported to occur during
long-term cold storage of fruits such as mangoes (Zhao et al., 2009), oranges (Huang
et al., 2008), and pawpaws (Galli et al., 2009). High AA levels in some apple culti-
vars have been linked to the high GSH levels, resulting in better storage properties
248 Advances in Postharvest Fruit and Vegetable Technology

(Davey and Keulemans, 2004). Apple cultivars such as “Greenstar” and “Braeburn”
were able to maintain or actually slightly increase GSH levels under both cold stor-
age and during shelf-life, and could retain quality for up to six months at 1°C (Davey
and Keulemans, 2004). A comprehensive study on the changes in different antioxi-
dants in apple fruit during cold storage revealed that GSH levels were significantly
higher in the fruit skin after 45 days of storage in air and then declined after 90 days
to harvest levels (Łata, 2008). The study supports the notion that concentration of
GSH increases in response to chilling-related stress as a mechanism for acclimati-
zation, but prolonged storage duration may result in decrease in its concentration
to original levels or below. At the same time, the increase in GSSG concentration
has been reported to cause a decrease in the ratio of GSH:GSSG, which has been
considered to be a marker of oxidative stress in the tissue (Tausz et al., 2004). The
decrease in glutathione concentration has been associated with internal browning in
methyl bromide-fumigated “Thompson Seedless” grapes (Liyanage et al., 1993) and
methyl-iodide-induced phytotoxicity in lemons (Ryan et  al., 2007). These studies
also reflect that most changes in different antioxidant compounds, including GSH,
are cultivar-specific. The role of GSH as an antioxidant has not been widely studied
in fruits. The concentrations of other antioxidant compounds such as phenolics and
flavonoids are strongly influenced by the temperature during and duration of stor-
age. Strawberry fruit stored at 10°C or 5°C showed higher antioxidant capacity, total
phenolics, and anthocyanins, than those stored at 0°C (Ayala-Zavala et al., 2004).
Strawberries stored at 0°C retained an acceptable overall quality for the longest stor-
age duration; however, berries stored at temperatures higher than 0°C showed higher
content of aroma compounds and antioxidant capacity during the postharvest period.

11.4.4 Storage Atmospheres
Storage atmospheres containing low O2 and/or high CO2 can retard the fruit metabo-
lism through reduced rates of respiration and ethylene production. The modification
of storage atmospheres also provides other benefits such as alleviation of certain
physiological disorders, and suppression of microbial and insect activity. The pres-
ence of low O2 atmospheres surrounding the fruit can decrease inter- and intra-cel-
lular O2 levels in the flesh tissue (Whitaker, 2004). Therefore, there is a potential for
reducing the rates of oxidative processes in the fruit. Both beneficial and detrimental
effects can be expected from storage under controlled/modified atmosphere (CA/
MA) conditions, depending on the concentrations of gases, the physiological matu-
rity of fruit and the preharvest environmental conditions. Control of superficial scald
in apples is a classic example of the suppression of oxidative reactions in the fruit by
CA/MA storage. Low O2 atmospheres have been known to reduce the incidence and
severity of superficial scald in apples (Patterson and Workman, 1962). The storage of
“Starkrimson Delicious” apples in atmospheres containing 0.7% O2 for 6–8 months,
markedly suppressed incidence of scald compared with air-storage (Lau et al., 1998).
“Braeburn” fruit held in atmospheres containing 1.2 or 1.5% O2 + 1.0 or 1.2% CO2
for six months had significantly less core-browning and superficial scald than fruit
held in air for the same period (Lau, 1998). However, CA-stored fruit were highly
susceptible to Braeburn browning disorder (BBD) and internal cavities (IC) after
Postharvest Oxidative Stress in Fresh Fruits 249

cool growing seasons. And the susceptibility of fruit to BBD and IC was greatest in
late-harvested fruit stored in 3.0% CO2 and 1.5% O2. Therefore, seasonal environ-
mental conditions, coupled with the stage of fruit maturity, can override the ben-
efits of low O2 storage atmospheres in providing protection against superficial scald
(Emongor et al., 1994; Lau, 1998). CA storage in combination with postharvest treat-
ment with an antioxidant such as diphenylamine (DPA) or ethylene action inhibitor,
1-MCP, can reduce the incidence of scald in apples and pears (Rizzolo et al., 2005;
Watkins et al., 2000). These studies suggest that CA storage alone cannot provide
protection against the incidence of scald in apple and pear, without the supplementa-
tion of a postharvest treatment with an antioxidant or 1-MCP.
The role of high CO2 during CA storage in development of postharvest oxidative
stress has also been studied in fruits. “Pink Lady”™ apples held in CA containing
1.5 kPa O2 and 5 kPa CO2 accumulated more H2O2 than those stored in air, indicat-
ing stress from the high-CO2 concentrations in storage and causing flesh browning
symptoms (De Castro et al., 2008). A direct comparison of the effects of CO2 con-
centration in CA on the oxidative stress during storage of “Blanquilla” pears was
investigated (Larrigaudière et al., 2001b). During six months of storage in regular air
or CA containing either 2% O2 + 0.7% CO2, or 2% O2 + 5% CO2, the incidence of
core-browning only occurred in pears stored at 5% CO2 which was associated with
the accumulation of lipid peroxidation products (ethane and TBARS) and lowest
concentration of AA after six months of storage. The effectiveness of the antioxida-
tive defense system might have decreased, causing lipid peroxidation and finally
browning. The study suggested that oxidative damage was involved in the CO2-
related physiological damage occurring during CA-storage in pears (Larrigaudière
et al., 2001b).
It is believed that significant changes in the antioxidant metabolites and enzymes
occur during the initial stages of CA-storage of apples and pears, which subsequently
affect the long-term storage behavior of fruit. Some of these changes may have det-
rimental effects on fruit quality as reported in “Conference” pears. “Conference”
pears, subjected to CA containing 2% O2 and 5% CO2 or regular air for 21 days
at −1°C, showed a rapid decrease in total ascorbate content and increase in DHA
under CA conditions (Larrigaudière et al., 2001a). A sharp increase in the activities
of SOD, APX, GR, and LOX was coupled with the accumulation of H2O2 and lipid
peroxidation products, when the fruit were exposed to CA. These changes marked
a significant amount of oxidative stress in “Conference” pears exposed to CA con-
taining 5% CO2, which can further lead to development of core-browning during
extended storage (Larrigaudière et al., 2001a). It is generally accepted that decrease
in AA concentration caused by late-harvesting and 5% CO2 concentration in the
storage atmosphere are mainly associated with the appearance of brown heart in
“Conference” pears (Zerbini et al., 2002).
A study on apple cultivars, “Šampion,” “Jonagold,” “Topaz,” “Sawa” and the
clone U 633, showed that the pool of antioxidants such as AA, thiols, and phenolic
compounds, in the skin tissues of fruit, showed a pronounced increase under CA
conditions (1.5% O2 + 1.5% CO2) during the initial 45 days of storage and should
be considered as an acclimation response (Łata, 2008). After 90 days of storage,
the antioxidant status was kept more efficiently in the CA as compared to storage
250 Advances in Postharvest Fruit and Vegetable Technology

in regular air. Cranberries (Vaccinium macrocarpon Aiton) could be stored for two
months at 3°C under storage atmosphere containing 30% CO2 + 21% O2 (Gunes
et  al., 2002). The storage atmosphere did not affect the content of total phenolics
or flavonoids. However, the total antioxidant activity of the fruit increased over-
all by about 45% in fruits stored in air, while this increase was prevented by stor-
age in atmospheres containing 30% CO2 + 21% O2. Longan (Dimocarpus longan
Lour.) fruit stored in a 5% O2 atmosphere (balance N2) for six days at 28°C markedly
delayed pericarp browning in association with maintenance of high total phenolic
content and reduced activities of polyphenol oxidase (PPO), POD and phenylalanine
ammonia lyase (Cheng et  al., 2009). Moreover, the fruit stored in a 5% O2 atmo-
sphere exhibited a lower relative leakage rate and pulp breakdown and higher DPPH
radical scavenging activity than fruit stored in air.
The short-term exposure to MA has also been reported to be beneficial for inhib-
iting lipid peroxidation and enhancing the antioxidant potential of fruit. Postharvest
exposure of kiwifruit cv. “Xuxiang” to pure N2 gas for 6 h maintained a high level of
firmness within 14 days of storage at 1 ± 1°C and reduced the decrease in the firm-
ness during shelf-life (Song et al., 2009). A similar study was conducted on loquat, in
which fruit exposed to 100% N2 for 6 h could retain their quality during 35 days stor-
age at 5°C (Gao et al., 2009). In kiwifruit and loquat, the short-term anoxic treatment
reduced the increases in membrane permeability and lipid peroxidation, delayed the
increases in both O2− production rate and H2O2 content, increased activities of SOD
and POD, but reduced LOX activity throughout storage period (Gao et  al., 2009;
Song et  al., 2009). These studies show that the beneficial effects obtained from
anoxic treatment could be due to reduced lipid peroxidation, enhanced antioxidant
ability and membrane integrity maintenance.
The nonenzymatic and enzymatic antioxidants in mangoes have also been stud-
ied with respect to CA storage (Kim et al., 2007; Niranjana et al., 2009). According
to Kim et  al. (2007), gallic acid, total hydrolysable tannins, total soluble pheno-
lics, and antioxidant capacity significantly decreased throughout mango fruit ripen-
ing from mature-green to full-ripe stages, but CA-storage can possibly delay the
decrease through delayed fruit ripening (Kim et  al., 2007). Higher concentration
of phenolics compounds and activities of CAT and POD and lower levels of carot-
enoids were found in “Alphonso” mangoes stored in CA containing 5% O2 and 5%
CO2 compared to those kept in regular air after 45 days storage at 8°C, which could
be attributed to the effect of CA on fruit ripening. CA-storage was also effective to
alleviate symptoms of chilling-related injuries in mangoes (Niranjana et al., 2009).
Peach fruit “cv. Okubao” stored at 0°C for 60 days under CA of 5% O2 + 5% CO2,
or CA with high O2 concentration 70% O2 + 0% CO2 for 15 days, then in CA with
5% O2 + 5% CO2 showed reduced chilling-related injuries compared to those held
in regular air (Wang et al., 2005). CA-storage delayed the reduction in activities of
SOD, CAT, and POD, which have been associated with the alleviation of chilling-
related injuries in peaches. High O2 treatment also induced the activities of SOD
and CAT, but no significant effect on alleviating chilling-related injuries was found
compared to CA-storage. The extent of lipid peroxidation was greatly reduced under
CA conditions, resulting in the maintenance of membrane integrity (Wang et  al.,
2005). A recent study showed that storage of Japanese plums “cv. Blackamber”
Postharvest Oxidative Stress in Fresh Fruits 251

in CA (1 or 2.5% O2 and 3% CO2) was favorable for the glutathione pool and the
enzymes related to restoration of glutathione in the AA–GSH cycle, but the effects
of CA on the ascorbate pool were not favorable (Singh and Singh, 2013a). The enzy-
matic and nonenzymatic antioxidative systems were efficiently operating under low
O2 atmospheres to scavenge the ROS produced in plums in the response to long-term
chilling and gaseous stresses. CA conditions appeared to be limiting the oxidative
processes to some extent, resulting in reduced oxidative damage in plums (Singh and
Singh, 2013a).
Posthypoxic and/or postanoxic conditions are among the stresses in which ROS
are implicated as the principle cause of injury. When aerobic conditions are reestab-
lished, a burst of ROS takes place, resulting in posthypoxic or postanoxic injury to
the tissues. A study was conducted on the monitoring of gene expression of enzymes
involved in ascorbate biosynthesis, oxidation, and recycling in tomato, in response to
hypoxia, and postanoxic stress (Ioannidi et al., 2009). To create hypoxic conditions,
mature green tomato fruit were subjected to atmospheres containing 0%, 0.5%, and
3% O2 (balance N2) for 1, 3, 6, 12, 24, 48, and 72 h at 22°C. For testing posthypoxic
stress, mature green fruit were subjected to 100% N2 for 48 h and then were exposed
to air. Postanoxic stress caused mature green tomatoes to respond by inducing the
transcript accumulation of all AA biosynthetic genes as early as 3–6 h after return to
air, coinciding with elevated levels of AA. Similarly, enzymes involved in AA recy-
cling responded to postanoxic stress by increasing their mRNA steady-state levels
upon return to air. This was an indication of the magnitude of the oxidative damage
and the crucial role of AA in scavenging ROS under these conditions. The induction
of AA recycling genes suggest that this massive activation of transcription is needed
to increase the reduced AA pool in order to compensate for the oxidative stress.
A study on the effects of anaerobic stress (exposure to N2 atmospheres for 24 h)
on the proteome of mandarins and grapefruit revealed that the majority of the identi-
fied citrus anaerobic proteins (ANPs) in both mandarins (eight out of 10 proteins)
and grapefruit (five out of nine proteins) were stress-related proteins, such as SOD,
APX and LOX (Shi et al., 2008). The activation of SOD and APX in response to
anaerobiosis might be crucial to preserve the redox status of the cells. Furthermore,
LOX is one of the main enzymes known to be involved in the generation of ROS
and, thus, the observed suppression of the LOX protein in mandarin flavedo may
be part of the cellular efforts to cope with the undesired accumulation of ROS. The
increase in the abundance of two oxidoreductases—zinc oxidoreductase and quinine
oxidoreductase—in flavedo may contribute to the elevated anaerobic stress tolerance
of “StarRuby” grapefruit (Shi et al., 2008).
It is therefore evident that storage of fruit under optimal CA conditions can poten-
tially maintain or slightly increase the antioxidant potential of the fruit. The short-
term hypoxia or anoxia can also stimulate the antioxidant defense system in the
fruit. But, there is absolute lack of information on the transcript levels of various
antioxidant enzymes involved in ROS scavenging and AA–GSH cycle in fruit sub-
jected to CA/MA. Most studies have been restricted to measuring the activities of
SOD, CAT, and POD and lipid peroxidation. The emerging tools of proteomics and
metabolomics have provided great insight into the development of anaerobic stress
in mandarin and grapefruit (Shi et al., 2008). The application of these tools has also
252 Advances in Postharvest Fruit and Vegetable Technology

been extended to study chilling-related injuries in peaches (Dagar et al., 2010). There
is vast scope for research in this area to explicate the processes involved in develop-
ing oxidative stress leading to physiological disorders in various fruits.

11.4.5 Postharvest Treatments
Postharvest oxidative stress has been associated with the enhanced rates of ripening
and senescence, the development of physiological disorders, and reduction in nutri-
tional quality of fruits (Hodges et al., 2004). Therefore, the regulation of oxidative
stress can be important to improving the shelf-life and quality maintenance during
postharvest handling, storage and distribution of fruits (Toivonen, 2003b). Postharvest
treatments to control oxidative stress-related injury in fruits involve two approaches:
(1) the use of antioxidant dips and coatings to directly prevent oxidation reactions,
and (2) postharvest treatments, such as low or high temperature, exposure to CA/MA
and growth regulators, to enhance endogenous resistance or tolerance to oxidative
stress. The postharvest treatments such as low-temperature conditioning (cold shock
treatments), heat treatments, and antioxidant dips, etc., also play an important role in
strengthening the antioxidant system of fruit (Toivonen, 2003b). The role of storage-
temperature, storage-duration and CA/MA in alleviating oxidative stress in fruits has
been reviewed in previous sections. In this section, the role of postharvest treatments
with ethylene action inhibitor, 1-MCP, and putative ethylene biosynthesis inhibitor,
nitric oxide, in relation to oxidative stress in fruits will be discussed.

11.4.5.1 1-Methylcyclopropene
Among different strategies to control ethylene, the postharvest exposure of fresh
produce to 1-methylcyclopropene (1-MCP) has emerged as the greatest tool of com-
mercial importance all over the world (Blankenship and Dole, 2003; Watkins, 2006).
The major beneficial effects of 1-MCP in fresh horticultural produce are discussed
in greater detail in Chapter 6. In this section, the effects of 1-MCP on the oxidative
behavior of fruits will be reviewed. Studies have shown that 1-MCP treatment can
reduce the lipid peroxidation in different fruits during postharvest storage and shelf-
life. The inhibition of accumulation of lipid peroxidation products in the skin and
flesh tissues of various fruits such as apples, pears, loquats, and mangoes (Cao et al.,
2009; Singh and Dwivedi, 2008; Vilaplana et al., 2006), indicates a positive role for
1-MCP in alleviation of oxidative stress and maintenance of membrane integrity. The
maintenance of membrane structural integrity has been demonstrated by the reduced
ion leakage from the skin or flesh tissue of 1-MCP-treated fruits such as apricot,
lychee (litchi), loquat, and pear (Cao et al., 2009; Egea et al., 2010; Larrigaudière
et  al., 2004; Sivakumar and Korsten, 2010). 1-MCP treatment has been shown to
affect the activities of primary antioxidant enzymes, SOD, CAT, and POD. A sig-
nificant increase in the activities of SOD, CAT, POD, and APX has been observed
in flesh tissues of fruits, such as apricot, loquat, mangoes, and pears, in response to
1-MCP treatment which might have helped to regulate the concentration of ROS in
the fruit tissue, resulting in lesser oxidative damage. Studies have also shown that
the concentrations of ROS were significantly lower in the 1-MCP-treated fruit com-
pared to untreated control (Cao et al., 2009; Larrigaudière et al., 2008; Singh and
Postharvest Oxidative Stress in Fresh Fruits 253

Dwivedi, 2008; Vilaplana et al., 2006; Wang et al., 2009). There are contradictory
reports on the effects of 1-MCP on the nonenzymatic antioxidants and antioxidant
capacity of fruits. 1-MCP treatment has been shown to reduce the levels of AA in the
flesh tissues of apples and pears (Larrigaudière et al., 2004; Vilaplana et al., 2006).
Another study showed that there was no consistent effect on the concentrations of
AA and GSH in 1-MCP-treated pears (Silva et al., 2010). The antioxidant capacity
in apples has been reported to increase in the skin tissue (Larrigaudière et al., 2004;
MacLean et al., 2006; Shaham et al., 2003; Vilaplana et al., 2006) and remain unaf-
fected in the flesh tissue (Vilaplana et al., 2006). The beneficial effects of 1-MCP
in retention of AA in the fruit have been mainly attributed to the retardation of
fruit-ripening and senescence (Egea et al., 2010; Sivakumar and Korsten, 2010). The
stimulation of phenylpropanoid pathway by 1-MCP treatment has been attributed to
the increase or maintenance of phenolics and flavonoids in the skin or flesh tissue
of fruits.
The effects of 1-MCP are influenced by the fruit cultivar, the 1-MCP dose, expo-
sure duration, and postharvest conditions. However, the response of the antioxidant
system to 1-MCP treatment has been tissue-specific in fruits. For instance, 1-MCP
treatment of “Golden Smoothee” apples increased the susceptibility of skin tissue to
browning by increased activity of PPO and POD (Larrigaudière et al., 2008), while
it was beneficial for the flesh tissue due to decreased lipid peroxidation and lower
accumulation of H2O2 (Vilaplana et al., 2006). Total phenolics in “Empire” apples
showed a significant increase in the skin, but a decrease in the flesh tissue during
cold storage for five months (Fawbush et al., 2009). The control of superficial scald
in “Granny Smith” apples has been attributed to the increase in lipid soluble anti-
oxidants in the skin of 1-MCP-treated fruit, while activities of CAT and APX were
suppressed by 1-MCP treatment (Shaham et al., 2003).
The exposure of fruit to higher doses of 1-MCP has some detrimental effects
on the antioxidant system of fruit. In strawberries, exposure to 1 µL L −1 1-MCP
increased the susceptibility to disease through reduced activity of PAL and lower
concentration of phenolics (Jiang et al., 2001). Similarly, in limes, the higher dose of
1 µL L −1 1-MCP increased the activity of POD, enhanced the skin-yellowing as com-
pared to lower doses, which had an inhibitory effect on the loss of chlorophyll (Win
et al., 2006). The impact of postharvest conditions on the efficacy of 1-MCP in influ-
encing the antioxidant potential of fruit has been demonstrated in “Sunrise” apples
(Qiu et al., 2009). The study showed that the phenolics fraction of the flesh tissue
digestate showed higher Folin Ciocalteu reaction, reducing capacity in response to
1-MCP treatment, only if the fruit were stored at ≥15°C for three weeks. The effects
of 1-MCP treatment on the retention of nonenzymatic antioxidants during storage
of “Empire” apples were also influenced by the storage atmosphere, but most of the
results were inconsistent (Fawbush et al., 2009). The literature indicates the role of
1-MCP in influencing the oxidative processes involved in ripening, senescence and
physiological disorder development.

11.4.5.2  Nitric Oxide

NO is an important signaling molecule, which plays an important role in ROS metab-
olism during normal and stress conditions in plants (Lamattina et al., 2003). NO is
254 Advances in Postharvest Fruit and Vegetable Technology

itself a free radical molecule with very high reactivity. The ability of NO to regulate
the levels and toxicity of ROS imparts it a “cytoprotective” role. Oxidative stress,
which is provoked by increased concentrations of O2− , H2O2, and alkyl peroxides,
can therefore be alleviated through protective effects of NO (Beligni et  al., 2002).
Additionally, NO itself possesses antioxidant properties (Beligni et  al., 2002). The
reaction of NO with lipid alcoxyl (LO·) and peroxyl (LOO·) radicals is rapid and
beneficial to prevent the propagation of ROS mediated lipid peroxidation. The inter-
action between NO and other cellular antioxidants provides indirect protection from
ROS damage. The toxic effects of NO are generated when it reacts with O2− to form
peroxynitrites (ONOO−) (Lamattina et al., 2003). Peroxynitrites can further react with
thiol groups of proteins and polyunsaturated radicals of fatty acid lipids of membrane,
causing serious damage to proteins, lipids, and DNA, and is also capable of generat-
ing the most damaging hydroxyl radicals through its protonation. Therefore, NO can
be cytotoxic and cytoprotective, depending on the local concentration of NO as an
effect of the rate of synthesis, translocation, effectiveness of removal of this reactive
nitrogen species, as well as its ability to directly interact with other molecules and
signals (Arasimowicz and Floryszak-Wieczorek, 2007; Beligni and Lamattina, 1999).
The role of NO in delaying fruit-ripening and senescence has been established
and discussed in more detail in Chapter 9. NO is thought to be involved in interfering
ethylene biosynthetic pathway in fruit (Lesham and Wills, 1998; Singh et al., 2009;
Zhu et al., 2006, 2008), which helps to inhibit ethylene-dependent responses in the
fruit. In the past few years, the role of NO in the alleviation of postharvest oxidative
stress has been reported in fruits such as kiwifruit, longan, peach, and tomato (Duan
et al., 2007; Zhu et al., 2006, 2008). The concentrations of NO that were higher than
optimum increased the oxidative stress in the fruit tissue as shown by increased lipid
peroxidation and accumulation of ROS (Zhu et al., 2008). Therefore, the cytoprotec-
tive effects of NO can only be obtained at certain concentrations of NO, which are
required to be optimized for different fruits. In general, the postharvest treatment of
fruit with either donor-compounds of NO or NO gas, resulted in decreased levels of
lipid peroxidation, reduced accumulation of superoxides and H2O2, and increased
activities of antioxidant enzymes favorable for delay in initiation and reduction of
oxidative stress (Duan et  al., 2007; Zhu et  al., 2006, 2008). These studies dem-
onstrate that NO is a powerful tool to regulate the oxidative stress in fresh fruits,
depending upon the concentration and stage of application of NO.

11.5 CONCLUSIONS
The process of senescence in a plant or plant organs such as fruit, involves vari-
ous degenerative changes in DNA, proteins and lipids, caused by oxidative stress.
The oxidative stress develops when the well-regulated balance between prooxidants
and antioxidants is disturbed, in favor of the prooxidants (Apel and Hirt, 2004).
The ROS are produced as byproducts of normal metabolism for their role in signal
transduction, but their proliferation in the cell can cause oxidative damage to the
macromolecules (DNA, proteins, and lipids). The concept of oxidative stress has
emerged to a great extent to explain the anomalies in development of various chronic
diseases and ageing of humans and animals. The role of oxidative stress in plants,
Postharvest Oxidative Stress in Fresh Fruits 255

due to environmental perturbations, became of significant interest to researchers

in the middle to late 1980s (Hodges et  al., 2004). However, the research on post-
harvest stress physiology in relation to oxidative stress gained impetus in the late
1990s. Postharvest procedures adopted to extend the potential storage or market-life
of a fruit also act as stress factors (Toivonen, 2004). The development of oxidative
stress in the fruit tissue has been demonstrated in response to the prolonged stress
conditions and inherent metabolic changes. Postharvest oxidative stress has been
associated with processes of fruit-ripening, senescence, and physiological disorders.
Several factors have been proposed to influence the occurrence and severity of post-
harvest oxidative stress in fruits, including: genotype; harvest maturity; storage con-
ditions; storage duration; and growth regulators, including ethylene and polyamines.
Postharvest oxidative stress has been associated with the development of serious
physiological disorders such as chilling-related injury in citrus (Sala, 1998), super-
ficial scald in apples (Barden and Bramlage, 1994; Whitaker, 2004), and internal
browning in apples (Toivonen, 2003b), pears (Veltman et al., 1999, 2000) and plums
(Singh and Singh 2012a,b). Several strategies have been developed to delay the devel-
opment of oxidative stress, either through enrichment of antioxidants (e.g., antioxi-
dant dip treatment in apples to prevent scald) or through activation and sustenance of
antioxidative systems in fruits, to cope with the increasing ROS production during
postharvest handling and storage. The understanding of oxidative behavior of fruits
to various preharvest and postharvest factors has led to minimizing the detrimental
effects of oxidative stress on fruit quality and shelf-life. Oxidative stress measure-
ments, such as lipid peroxidation and activities of antioxidant enzymes and nonen-
zymatic antioxidants, are essential to understand the physiology and biochemistry of
postharvest processes. The oxidative stress indicators may be developed as biomark-
ers and tools for predicting and monitoring the postharvest behavior of fruits during
the supply chain. However, the complexity of oxidative and antioxidative systems in
multiple fruit species and/or cultivars is a great challenge to develop and evaluate the
robustness of these biomarkers. The oxidative stress theory holds potential to screen
germplasm for desirable postharvest traits and also to evaluate the efficacy of various
postharvest treatments or procedures aimed to enhance storage stability of fruits.

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 12 Advances in Postharvest
Maintenance of Flavor
and Phytochemicals
Jun Song

CONTENTS
12.1 Introduction................................................................................................... 261
12.2 Flavor............................................................................................................. 262
12.2.1 Flavor and Flavor Perception............................................................. 262
12.2.2 Biosynthesis and Regulations of Volatile Aroma Compounds..........264
12.2.3 Advances in Genetic Information on Flavor Compounds in Fruit......268
12.2.4 Postharvest Treatments’ Influence on Flavor Quality....................... 270
12.3 Bioactive Compounds and Phytochemicals................................................... 270
12.3.1 Genetic Factors Affecting Bioactive Compounds and
Phytochemicals.................................................................................. 272
12.3.2 Postharvest Storage Effects on Bioactive Compounds and
Phytochemicals.................................................................................. 273
12.4 Future Research Perspectives........................................................................ 275
Acknowledgment.................................................................................................... 278
References............................................................................................................... 278

12.1 INTRODUCTION
Fruits and vegetables play an important role in the human diet. Consumption of fresh
fruit is increasing, as consumers become more aware of the importance of food con-
sumption in relation to human health and its role in disease prevention. In addition
to their nutritional benefits, fruits are recognized for their delicious flavor, which is
important for enjoyment of food. The quality of fresh fruit and vegetables comprises
multiple aspects, including texture, appearance, color, flavor, and nutrition (Defilippi
et al., 2004). Among the quality indices, flavor may be one of the most important
traits; it is characteristically diverse, and brings enjoyment to the consumption of
fruit and vegetables. The growing understanding of flavor as an important quality
trait is reflected in the increased research efforts on the control and maintenance of
the quality of flavor in many fruit and vegetables. Discussing the trends in research
on flavor, Klee (2010) pointed out that most new technology developments have
focused more on production, yield, decay resistance, and appearance, rather than on
flavor (Klee, 2010). Improving the flavor properties of fresh fruit would add value,

261
262 Advances in Postharvest Fruit and Vegetable Technology

increase consumption, add health benefits by increasing consumption, and create

new markets for these commodities. It is also needed to satisfy consumer demands
(Kader, 2004; Morris and Sands, 2006). Meanwhile, fruits and vegetables offer a
wide variety of bioactive compounds such as flavonoids, phenolics, anthocyanins,
phenolic acids, stilbenes, and tannins, as well as nutritive compounds such as essen-
tial oils, carotenoids, vitamins, and minerals. Many of these compounds have potent
antioxidant, anticancer, antimutagenic, antimicrobial, anti-inflammatory, and anti-
neurodegenerative properties, both in vitro and in vivo. There has been a clear rec-
ognition for the need to shift from judging quality based on appearance, to judging
quality based on flavor and nutritional value, in the postharvest life of fruits and
vegetables (Kader, 2004; Klee, 2010). Therefore, it is worthwhile to look at these
unique quality characteristics, and assess the literatures from a postharvest perspec-
tive, to reveal control and regulatory mechanisms that can be used to maximize
the eating quality of fresh produce. As indicated in other sections of this book, the
main focus will be on recently reported studies on flavor, and developed technolo-
gies and systems that will assist the horticulture industry to be more environmentally
sustainable, and economically competitive in minimizing postharvest quality loss,
and to generate products that are appealing and acceptable to consumers. It is also
important to point out that many factors, from production to consumption, such as
preharvest genetics, environment, culture practices, fertilizers, irrigation, and stress
conditions, will influence postharvest flavor and phytochemical quality. It is not pos-
sible to cover each individual commodity as well as each reported study; therefore,
only selected commodities will be discussed.

12.2 FLAVOR
12.2.1 Flavor and Flavor Perception
Flavor has been used as a term to describe the consumer’s perception (eating experi-
ence) of a product. It integrates multiple indices, including taste (sweetness and acid-
ity), and smell (aroma/volatiles). Sweetness and acidity are important flavor factors
that have been long recognized. However, human perception of volatile compounds
is much more complicated, and determined by at least three factors: (a) fruit vola-
tile concentration; (b) aroma perception threshold; and (c) human aroma receptors.
Great progress has been made to characterize the production of fruit volatiles, in the
past twenty years. However, the science around flavor has been found to be more
complicated than most people first perceived. On the chemistry side, in addition to
identification of sugars and acids, over 300 chemical compounds have been reported
and identified as aroma/volatiles compounds in many fruits and vegetables. These
compounds include esters, acids, alcohols, aldehydes, ketones, and terpenes. The
diverse composition of volatiles among commodities indicates the complex blend
of flavor and complex pathways leading to volatile biosynthesis, and regulation in
each type of fruit and vegetable. Despite the low concentration of these compounds
present in nature, in comparison with sugars and acids, most of these compounds
have very different aroma thresholds, and therefore, some may still be important
contributors to the overall flavor perception, even at trace concentration. As a typical
Advances in Postharvest Maintenance of Flavor and Phytochemicals 263

example, aroma compounds in tomatoes have been intensively studied, but among
the hundreds of compounds identified, only 7 compounds are believed to be critical
to aroma/flavor perception (Buttery et al., 1989). New research has demonstrated,
however, that even the aroma threshold cannot solely be representative of human
perception. Some of the most abundant volatiles do not contribute to consumer
liking, whereas other, less abundant ones, do. For example, C6 volatiles (hexanal,
cis-3 hexenal, trans-2-hexenal, and hexyl alcohol) had been identified as among the
impact volatile compounds with high abundance, contributing to the flavor of the
tomato fruit, before it was proved that, even if the production of these compounds
was reduced significantly, it has no significant impact on consumer liking or flavor
perception (Tieman et al., 2012). New findings also point to the fact that aroma vola-
tiles contribute to perceived sweetness, independent of sugar concentration, suggest-
ing a novel way to increase perception of sweetness without adding sugar (Tieman
et al., 2012). A positive correlation of overall flavor with total titratable acidity, and
total soluble solids, also indicates that these two components play an important role
in determining overall flavor. Subjectively measured traits, including fruity odor
and fruity flavor, had positive correlations with overall flavor (Panthee et al., 2013).
However, there was no correlation between fruit firmness and overall flavor. This
indicated that sweetness can be a target for breeding programs aiming to improve
the overall flavor of the tomato fruit. It implies that breeders can simply focus on
selecting tomato accessions for a few traits, including sweet taste, juiciness, and juicy
texture (Panthee et al., 2013).
For other fruit, depending on the flavor characteristic, there may be different targets.
For example, it is well known that many fruits produce significant amount of esters,
which have “fruity” and “sweet” flavor characteristics. Due to low aroma thresh-
olds, these compounds contribute significantly to the overall “fruity” flavor of ripe
and mature fruit. The divergent blends of these ester compounds result in very dif-
ferent flavor perceptions by consumers of these fruit. Butyl acetate, hexyl acetate,
and 2-methylbutyl acetate are the most important volatiles present in ripe fruit—as
found in “Golden Delicious” apples (Song and Bangerth, 1996). While, hexyl hex-
anoate and hexyl butanoate may present a “senescent” and “over-ripe” flavor to the
same apples, but at the late stage of fruit-ripening and senescence (Song and Forney,
2008). In a study investigating the effect of modified atmosphere packaging (MAP)
using ultra microperforated film (MP), on the sensory quality of apple slices, a sig-
nificantly higher mean score for fruity aroma, for the MAP packages from the south-
ern hemisphere, was directly attributable to the higher volatile concentrations for
straight-chain esters (Isaacson et al. 2006), estragole, total-volatiles, other-volatiles,
and terpene compounds (Cliff et al., 2010). Interestingly, there was higher perceived
sweetness of the fruit in the MP packages, compared to the steady-state atmosphere
packages. While this does not appear to be associated with changes in the soluble-
solids content, it may be the result of the psychological interaction and perception of
sweetness and fruitiness, as noted in the literature. In a model system with no sugar,
samples are perceived sweeter when fruity odors/aromas are present. Such associa-
tions are attributed to cross-modality (taste–smell) interactions particular for con-
gruent sensation. While the compounds involved do not physically or physiologically
interact, there is a strong and very real learned association, particularly for repeated
264 Advances in Postharvest Fruit and Vegetable Technology

exposure, sensations typically occurring with regard to maturing fruit (Cliff et al.,
2010). This research is the first of its type to document that higher volatile concen-
trations, associated with the MP films, translates to greater perceived fruitiness, and
more flavorful apple slices, as perceived by sensory panelists.
These new findings indicate the complex interaction of human flavor perception
and flavor chemistry. The application of aroma thresholds is dependent on different
volatile compounds and individual fruit, and there is no universal fit using aroma
thresholds to all cases of the flavor perceptions of consumers.

12.2.2 Biosynthesis and Regulations of Volatile Aroma Compounds

Despite intensive research for many years, biosynthetic pathways for most volatile
compounds in fruits and vegetables have not been elucidated. Traditionally, volatiles
can be classified as “primary” or “secondary,” indicating whether they were pres-
ent in intact fruit tissue, or produced as a result of tissue disruption (Dirinck et al.,
1989). For many fruits and vegetables, such as apples, bananas, melons, and straw-
berry, flavor impact volatiles can be considered as primary compounds; however, the
main flavor volatiles in tomatoes, cucumbers, and lemons are secondary volatiles.
Therefore, aroma perception of fresh produce can vary, depending on how aroma is
generated, and when the product is consumed. It is necessary, in every aroma study,
to clearly state the procedure for sample preparation and assessment. Aroma pro-
duced from an intact fruit relates to the consumer’s perception and judgment of the
product for being “ripe,” “unripe,” or “off-flavor,” while aroma volatiles produced
after tissue disruption may reflect the consumer’s perception of flavor during eating
and chewing. It is also important to pay attention to the effects of tissue disruption
on flavor generation. Due to enzyme activities, and nonenzyme chemical reactions,
volatiles generated after tissue disruption may be unstable, and prone to change, and
therefore, it is necessary to stabilize and standardize the volatile analysis procedure
for volatile research. Methods to stabilize volatiles involve using buffers and salts,
including calcium and sodium, which have been used on tomato samples (Buttery
et al., 1989). Although the metabolic pathways for volatile biosynthesis in most fruit
are not fully understood, it is known that more than 80% of the volatiles produced
by ripe apples are esters (Dirinck et al., 1989). Therefore, production of these com-
pounds has been the major focus of research in the past. The last enzyme that is
responsible for ester formation is alcohol acyltransferase (AAT, EC 2.3.1.84), which
combines alcohols and acyl CoAs to form esters. Other enzymes, such as lipoxygen-
ase (LOX, EC 1.13.11.12), alcohol dehydrogenase (ADH, EC 1.1.1.1.), and pyruvate
decarboxylase (PDC, EC 1.2.4.1), are also believed to be involved in the pathways
to provide aldehydes and alcohols (Dixon and Hewett, 2000; Fellman et al., 2000).
Using transgenic lines that block ethylene biosynthesis, AAT was found to be regu-
lated by ethylene, while ADH and LOX were unaffected by ethylene modulation.
Isoleucine, which is an important precursor for the branched esters of 2-methyl-
related compounds, increases in the peel, and is also regulated by ethylene (Defilippi
et al., 2004). Exogenous ethylene treatment of immature apple fruit induced fruit-
ripening and volatile production, with preference to branched esters (Song, 1994).
Advances in Postharvest Maintenance of Flavor and Phytochemicals 265

It was reported that more than 88 acyltransferase were found in Arabidopsis,

but a limited number of them have been characterized with a known biochemical
function (St-Pierre and De Luca, 2000). Cloning of MpAAT has been conducted
in “Royal Gala” apples. It was report that MpAAT is expressed in leaves, flowers,
and fruit. The MpAAT gene product (protein) has the AAT function, and can use a
wide range of substrates, including both straight chain (C3 –C10) and branched chain
alcohols. However, the binding of alcohol substrates is rate-limiting, compared with
the binding of CoA substrates. The preference of MpAAT1 for alcohol substrates is
dependent on substrate concentration, which determines the aroma profiles of apple
fruit (Souleyre et al., 2005). Another AAT gene, MdAAT2, was cloned in “Golden
Delicious” apples. It has low sequence identifiers, in comparison with other fruit
AATs. In contrast to other apple varieties, the MdAAT2 of “Golden Delicious” was
exclusively expressed in the fruit (Li et al., 2006). The MdAAT2 protein is about
47.9 kD, and is localized primarily in the fruit peel. Data also demonstrates that the
expression of the MdAAT2 protein is regulated at the transcription level, in the fruit
peel. MdAAT2 was inhibited by treatment with 1-methylcyclopropene (1-MCP), and
influenced by ethylene (Li et al., 2006). The expression levels of both MdAAT1 and
MdAAT2 increased with the progression of fruit-ripening, and correlated with the
total amount of esters detected in “Golden Delicious” and “Granny Smith” apple
cultivars (Zhu et  al., 2008). In particular, MdAAT1 and MdAAT2 coincide with
the genes described by Souleyre et al. (2005), and Li et al. (2006), respectively. As
with AAT in melons and strawberries, it was also revealed that substrate availabil-
ity is more important, than AAT activity, in determining ester formation in apples
(Beekwilder et al., 2004). Another study conducted on banana and strawberry fruit
found similar results—that the substrates determine the characteristic aroma profiles.
It has been reported that both the fatty acid and branched amino acids may
function as the precursors of volatile formation (Rowan et  al., 1999; Song and
Bangerth, 2003). The straight-chain volatile, with C2 , C4, and C6 carbons, may
be derived from fatty acids metabolism, to aldehyde, which is further converted
to alcohols by the ADH. It is widely assumed that lipoxygenases may contribute
to the breakdown of long-chain fatty acid to C6 aldehydes, which are converted to
alcohols by aldehyde dehydrogenase. Branched amino acids are important sub-
strates for branched chain volatiles such as 2-methylbutylactate or ethyl-2-methyl-
butanoate (Rowan et al., 1996).
The source of alcohols and aldehydes, leading to ester synthesis in apple fruit, is
not fully understood. LOX catalyzes the first step in the metabolic pathway to convert
the 18:2 and 18:3 fatty acids to C6 volatiles, including cis-3-hexenal, hexanal, and their
alcohols. Despite the possible role of LOX in tomatoes, it is interesting to note that
there is no close correlation between LOX activity and ester formation in “Golden
Delicious” apples at early and middle maturity harvests (Song, 1994). However, a
better relationship between LOX and ester formation can be seen in late harvested
fruit. Using multivariance analysis of the biosynthesis of volatile compounds, it was
concluded that LOX and PDC are responsible for the differential production volatiles
that occur in CA and regular air (RA) stored fruit, while no difference in AAT activ-
ity was found (Lara et al., 2006). Further research on fatty acid biosynthesis in apple
266 Advances in Postharvest Fruit and Vegetable Technology

fruit, with different harvest maturities, revealed that newly synthesized free fatty
acids may be used as precursors for aroma biosynthesis (Song and Bangerth, 2003).
Strawberry fruit produce more than 300 volatile aroma compounds, the major-
ity being esters, alcohols, aldehydes, terpenes, and acids. Total volatile production
significantly increases as fruit-ripening advances from white to pink, to red. This
increase of volatile compounds is closely related to total volatiles, esters, and acids,
but negatively related to alcohols, which are predominant in white fruit. Using a
quantitative proteomic approach, the increase of total volatile compounds and esters
is well correlated with the protein profiles associated with aroma production in
strawberry. It identified and quantified two AATs, two quinone oxidoreductases and
O-methyltransferase (Li et  al., 2013). The involvement of AAT in ester formation
of many fruit is well known (Pérez et  al., 2002). AAT is one of the most impor-
tant enzymes to catalyze the formation of esters in strawberry fruit during ripening
(Aharoni et al., 2000). At the proteomic level, two AAT proteins (AAT1 and AAT2)
increased more between the pink-to-red stage, than from the white-to-pink stage,
in both “Mira” and “Honeoye” strawberry fruits. This change confirmed the results
at the transcript level reported by Aharoni, that AAT expression is induced in fruit-
ripening (Aharoni et al., 2000). In addition, two pyruvate decarboxylases (PDC) in
both “Honeoye” and “Mira,” and acyl-CoA synthetase in “Mira,” were also found to
be significantly upregulated. These increases can be explained by the high demand
for acyl-CoA for ester and fatty acid biosynthesis during ripening. Proteomic work
provides evidence that PDC and AAT become more abundant concurrent with rapid
volatile production, in ripening strawberry fruit.
The furanones—4-hydroxy-2, 5 dimethyl (2,3H) (DMHF, furaneol), and
4-methyoxy-2, 5 dimethyl (2,3H) (DMMF, mesifurane)—have been recognized as
key volatile compounds in strawberry, and a group of 3(2H)–furanone compounds
have been identified in strawberry fruit (Ulrich et  al., 1997; Bood and Zabetakis,
2002). Using HS-SPME, DMMF was identified in both “Mira” and “Honeoye” cul-
tivars (Li et  al., 2013). DMMF has a signature olfactory characteristic of “sweet”
and “burnt sugar” notes, and its methylation is mediated by an O-methyltransferase
(FaOMT), whose activity increases during fruit-ripening (Lavid et al., 2002). The
formation of furanones is under the control of FaOMT, a gene that is responsible for
the variation of the natural mesifurane content in strawberries (Wein et al., 2002;
Zorrilla-Fontanesi et  al., 2012). In both “Honeoye” and “Mira,” it was found that
there was a significant increase in OMF protein, as fruit ripened from white to red,
and which coincided with the increase of DMMF concentration. In addition, two
quinone oxidoreductases were identified, which both increased during ripening. The
quinone oxidoreductase FaQR, has been shown to be strongly induced by ripening,
and to be auxin-dependent (Raab et al., 2006). This enzyme functions as an enone
oxidoreductase in the biosynthesis of DMMF, and is responsible for the conversion
of DMHF to DMMF. In anti-ACO line kiwi fruit, it increased 5-fold in expression,
and peaked at 168 h after ethylene treatment (Atkinson et al., 2011). These results
reveal the important role of quinone oxidoreductase in volatile biosynthesis of straw-
berry during fruit-ripening.
In addition to enzymes and substrates, other factors may also be equally impor-
tant. In early harvested immature fruit, fruit treated with 1-MCP, or fruit subjected
Advances in Postharvest Maintenance of Flavor and Phytochemicals 267

to long-term low oxygen storage, metabolic energy, such as ATP, may be limited, and
thus constrain volatile production. All of these conditions can reduce fruit respira-
tion, as well as rate of metabolism (Rudell et al., 2002). Saquet et al. (2003) postu-
lated that other cofactors, such as pyridine nucleotides (NADH and NADPH), are
not reduced in CA-stored fruit. Low rates of respiration decrease ATP as well as the
ATP:ADP ratio, which directly reduces fatty acid biosynthesis (Saquet et al., 2003).
Further study is needed to elucidate the control mechanisms of volatile biosynthesis
during fruit-ripening and senescence.
The branched amino acids leucine and isoleucine, are important substrates for
branched chain volatiles, such as 2-methylbutyl acetate, and ethyl-2-methylbutanoate
in apple, or 3-methylbutyl acetate in banana (Tressl and Drawert, 1973; Fellman
et al., 2000). Feeding studies show that this pathway may be present in many fruits,
such as apple, banana, and strawberry (Tressl and Drawert, 1973; Rowan et al., 1996;
Fellman et  al., 2000; Perez et  al., 2002). In melons, both fatty acid and branched
amino acid biosynthesis are important contributors to volatile formation. The amino
acids alanine, valine, leucine, iso-leucine, and methionine, increased during fruit-
ripening, in close association with volatile production, and are believed to supply the
carbon chains for four groups of esters, ethyl acetate, 2-methylpropyl, 2-methylbu-
tyl, and thioether ester, respectively (Wyllie et al., 1995; Wang et al., 1996). When
a comparison of amino acid content was made between the highly aromatic melon
“Makdimon” and the low-aroma melon “Alice,” no significant difference in amino
acids concentration was found. Therefore, the difference in volatile concentrations in
melon is not due to the availability of amino acid substrates, but rather is dependent
on other biosynthetic pathways (Wyllie et al., 1995).
Using tomatoes as another example, the analysis of “secondary” volatile com-
pounds become a more routine procedure (Baldwin et al., 2007). A recent report
indicated that several genes related to volatile biosynthesis in tomatoes have been
identified through the candidate gene approach, or screening for genes encod-
ing enzymes that might function in a given synthetic pathway (Klee and Tieman,
2013). However, the whole production process from selection of genetic materials,
preharvest practice, to postharvest techniques, will influence flavor and posthar-
vest quality.
Molecular biology tools need to be applied to better understand the biosynthesis
of aroma volatiles in fruit. Genomic tools, which have been developed on model sys-
tems, could be applied to fruit and vegetables. Although few genomes of fruits and
vegetables have been completely sequenced, many genomic tools that are available
today for Arabidopsis (Arabidopsis Information Resource), Solanaceae (Solanaceae
Genomic Network), and Rosaceae (Rosaceae Genome Database), are paving the
way for future research and development in genomics and proteomics. Apple has
more than 60,000 protein sequences reported in NCBI (http://www.ncbi.nlm.nih.
gov/protein, accessed on December 31, 2014). A high-quality draft of the complete
genome of pear (Pyrus × bretschneideri) reports 42,812 protein coding genes, with
ca. 28% encoding multiple isomers (Wu et  al., 2013); there were 46,518 protein
sequences reported for public use on (http://www.ncbi.nlm.nih.gov/protein, accessed
on December 31, 2014). Undoubtedly, the ultimate success of proteomic research is
dependent on the completion of genome sequences of fruits and vegetables, which
268 Advances in Postharvest Fruit and Vegetable Technology

will provide complete information about proteins, and their modifications, that can
be identified in biological studies.
Applying an expressed sequence tag (EST), frequency analysis with EST database
revealed that EST clusters from fruit-derived tissue show strong sequence homology,
with biochemically characterized enzymes, that are involved in ester biosynthesis,
including acyl-CoA dehydrogenase, acyl-CoA oxidase, enoyl-CoA hydratase, acyl
carrier proteins, malonyl-CoA:ACP transacylase, LOXs, 3-ketoacyl-CoA thiolase,
acyl-CoA synthetase, and acyl carrier proteins (Park et al., 2006). Two highly diver-
gent ADH genes (CmADH1 and CmADH2) are expressed in ripening melon fruit,
and have been cloned in melon (Cucumis melo var. Cantalipensis). These enzymes
are closely related to fruit ethylene production, and are inhibited by 1-MCP, indicating
that they are under control of ethylene. Sequence analysis indicated that CmADH1
has 83% homology with apple Md-ADH (Marinquez et al., 2006). These findings
add new clues for enzymes responsible for aroma volatile biosynthesis beyond AAT,
LOX, and fatty acid ß-oxidation, and may open new opportunities for aroma volatile
research to identify unknown pathways related to fruit volatile biosynthesis.

12.2.3  Advances in Genetic Information on Flavor Compounds in Fruit

In a review paper, Klee and Tieman (2013) discussed these two specific challenges
for flavor improvement from a breeder’s perspective:

• What are the important chemicals that contribute to consumer preference,

either positive or negative?
• What genes control the synthesis of these chemicals, and what are the pos-
sible alleles of the most important genes?

The answers to these questions will lead to the introduction of a series of genes into
the genome (Klee and Tieman, 2013). With the great progress in genome sequencing
of many fruit and vegetable species, full genomic information has been made avail-
able, not only to plant breeders, but also to chemists, biochemists, and physiologists,
to aid in the improvement of flavor quality. Genetic mapping of quantitative trait loci
(QTLs) involves identifying and determining the degree of association between cer-
tain traits and a set of genetic markers. A saturated genetic map, covering the entire
genome, is essential for accurate QTL identification. The identification of QTLs
linked to important traits in apple, such as disease resistance, tree growth, and fruit
quality, is still at an initial stage. A quantitative genetic analysis of traits associated
with apple fruit-flesh firmness, using a population derived from a “Prima” × “Fiesta”
cross, was conducted (King et al., 2000). QTLs accounting for differing degrees of
variation for firmness, stiffness, and a number of sensory attributes, were identified
on seven linkage groups (LG), with large effects on LG1, LG10, and LG16. Further
work extended the range of mechanical measurements to include compression, and
wedge fracture tests (King et al., 2000). The wedge fracture tests identified signifi-
cant QTLs on LG16 and LG1. The QTL on LG16 was located in the same region
as the QTL identified for certain sensory textural attributes, such as crispness and
juiciness.
Advances in Postharvest Maintenance of Flavor and Phytochemicals 269

Employing linkage mapping, and map calculation using a subset of 57 out of

the 86 individuals of the cross between “Fiesta” and “Discovery,” volatile organic
compounds from ripe apple fruit were analyzed with proton transfer reaction–mass
spectrometry (PTR–MS). Abundance of mass 61, which is a common fragment of
acetate esters, and the observed ratio between mass 43 and 61, are in excellent agree-
ment with that observed for acetate esters, indicating a possible link between ester
production and QTL (Zini et al., 2005). During further study on the same popula-
tions, a set of QTL associated with major volatile compounds was identified, and
confirmed across three different locations. The volatile organic compound (VOC)
groups showing higher values of heritability among locations were related to esters,
which is one of the most important compound classes for fruit aroma. In this specific
case, several masses enabled the detection of QTL, located in two regions of chro-
mosome 2, while another volatile peak, assigned to ethylene, was mapped on chro-
mosome 15 (Costa et al., 2013). Using the assembled “Golden Delicious” genome
database, 17 apple AAT members were targeted and annotated. When the MdAAT1
primers were used, a 468 bp nucleotide sequence was obtained for sequence com-
parisons among the 102 apple cultivars (Dunemann et  al., 2012). High sequence
similarity was observed among the 102 cultivars, with only four SNPs observed at
nt positions 62, 110, 425, and 459 bp, according to the position in the reference gene
sequence MDP0000637737 of “Golden Delicious.” These SNPs caused changes in
the amino acid sequence at positions 258 (I–T), 274 (C–Y), and 379 (V–A). The
association study revealed significant associations of individual SNPs and specific
haplotypes, with the trait “ester concentration.” These remarkable phenotypic differ-
ences observed between apple cultivars were clearly caused by the genotype, with
minimal influence of year of harvest, suggesting apple aroma as a highly heritable
trait (Dunemann et al., 2012). Even if consumer preference for the flavor of apple
varieties is highly subjective, ester-accentuated flavor types are especially success-
ful on markets worldwide. Therefore, the ester content of apple fruit is an important
goal in an apple cultivar breeding program. The use of marker-assisted selection
using functional markers for fruit flavor traits, including ester content, is assumed to
shorten the long traditional breeding activities.
Genetic investigation of LOX genes detected a total of 15 QTLs for eight volatiles
(esters and C6 aldehyde, hexanal) in apples, which were located on chromosomes
2, 7, 9, and 12. At least four genome regions were identified to be associated with
LOX candidate genes by QTL mapping. The QTLs associated with the MdLOX5
gene cluster on chromosomes 2 and 7, might be explained by the function of type 2
(13-LOX), while the association of QTL with the MdLOX7 cluster on chromosome
12, is more complex, and needs more functional study at the protein level (Vogt
et al., 2013). Unlike AAT, the genetic information with LOX may not link directly
to the esters production, since the direct substrates are not directly controlled by the
LOX. Ester formation may be regulated at the subsequent enzymatic levels (HPL and
ADH), and the availability of fatty acids may affect the production of LOX-derived
volatiles, masking other influence enzymes (Vogt et al., 2013).
Sweetness and acidity play an important role in fruit flavor. Fruit acidity is due to
the presence of organic acids, and malic and citric acids are the main acids found in
most ripe fruit (Seymour et al., 1993). A recent study, conducted using two half-sib
270 Advances in Postharvest Fruit and Vegetable Technology

populations GMAL 4595 [Royal Gala × PI (Plant Introduction) 613988] and GMAL
4590 of 438 trees, demonstrated that the Ma locus, which is controlling the malic
acid content in apples, is the primary genetic factor determining fruit titratable acid-
ity, and/or pH in both “Royal Gala” and the two Malus sieversii accessions PI 613988
and PI 613971 (Xu et al., 2012). In the “Golden Delicious” genome, the homologous
Ma region, defined by the five flanking markers, is no larger than 150 kb, and con-
tained 44 predicted genes. In addition, there are two minor QTL detected for fruit
TA and pH with M2 specific to Royal Gala, and M3 to PI 613988. With the QTL and
markers identified/developed, it might be possible to screen apple breeding popula-
tions at the seedling stage, to remove or select most of the targeted genotypes (Xu
et al., 2012). The eight new simple sequence repeat (SSR) markers developed would
be useful in marker-assisted breeding in apple. Construction of the fine map of the
Ma locus represents an important step forward in isolating the Ma gene. Those 44
predicted genes are largely hypothetical, and quite diverse, including 19 of hypo-
thetical proteins. Further study is required to confirm and reveal the identity of the
Ma gene (Xu et al., 2012).

12.2.4 Postharvest Treatments’ Influence on Flavor Quality

The effect of harvest maturity and postharvest treatments on volatile biosynthesis
and flavor in many fruits and vegetables, has been well documented (Mehinagic
et al., 2006; Song and Forney, 2008; Bennett, 2012).
When fresh-cut cantaloupe cubes were treated with 1.0 μL/L of 1-MCP for 24 h
at 5°C, and stored in air at 5°C for nine days, most quality attributes were unaf-
fected by the treatment with 1-MCP (Amaro et al., 2013). It preserved soluble solids,
total phenolics, total carotenoids, and β-carotene content, but significant softening
occurred. 1-MCP-treated fresh-cut cantaloupe accumulated higher levels of propyl
acetate, 2-methylbutyl acetate, methyl butanoate, methyl 2-methyl butanoate, methyl
hexanoate, 2-methylbutyl alcohol, and phenethyl alcohol, and lower levels of benzyl
alcohol and heptanal, than untreated controls, particularly those derived from the
amino acids isoleucine and phenylalanine, but had no significant effect on other
phytochemicals or quality attributes.

12.3  BIOACTIVE COMPOUNDS AND PHYTOCHEMICALS

It is well known that fruits and vegetables are major sources of nutrients and phy-
tochemicals for the human diet. Increasingly more epidemiological evidence has
become available for possible effects of specific compound(s) on human health and
disease prevention. This chapter will emphasize recent research on phytochemicals
and bioactive compounds in fruits and vegetables, with specific interest on postharvest
aspects. It is not intended to be a comprehensive coverage of all the research on bioac-
tive compounds. The main goal of this section is to provide updated information on
the effects of various factors such as genetics, environment, and postharvest storage,
on the bioactive compounds and phytochemicals. It is expected that new knowledge
of these factors can lead to multidisciplinary strategies to maximize the bioavailabil-
ity and health potential of fruit and vegetables, during postharvest handling.
Advances in Postharvest Maintenance of Flavor and Phytochemicals 271

For better understanding of the bioactive compounds and phytochemicals in

fruits and vegetables, it is necessary to outline the major groups and categories. In
general, bioactive compounds and phytochemicals can be divided into three catego-
ries: polyphenols, glucosinolates, and carotenoids. Under the polyphenols category,
further subgroups can be seen as flavonoids, phenolic acids, stilbenes, and lignans.
The well-known flavonoids groups in many fruits can further divided into isofla-
vones, flavones, flavonols, flavanols, anthocyanins, and flavanones. The abundance
of each group of compounds differs significantly in different fruits and vegetables.
For example, berry fruit contain significant amounts of flavonoids, which can be
seen as distinct blue or red in color. High bush blueberry (Vaccinium corymbosum)
and strawberry fruit contain large amounts of total phenolics (251–310 mg/100 g
and 222–225 mg/100 g, gallic acid equivalent, respectively), and total anthocya-
nins (92–129 mg/100 g, cyanidin 3-glucoside FW and 35.6 mg/100 g, pelargonidin
3-glucoside FW, respectively) (Aaby et al., 2007). The distribution of phenolic com-
pounds in tissues varies greatly in different parts of fruits and vegetables. Strawberry
fruit achenes contain about 14% higher levels of phenolics and 12% higher levels of
anthocyanin, when compared to the strawberry flesh (Aaby et al., 2005). The skin
of apples has a concentration of 5-caffeoylquinic acid and catechins higher than in
the cortex parenchyma. The levels of hydroxycinnamic acid and catechin in apples
and pears generally increase for a short time during initial development, and are sig-
nificantly high in the raw fruits (Boyer and Liu, 2004). In mature cherries, the phe-
nolic acids, such as derivatives of synaptic acid, are often localized in the epicarp.
In grape skin, the concentration of hydroxylcinnamoyl tartaric acid is several-fold
greater than in the pulp (Li et al., 2012).
Antioxidant capacity has been widely used to describe the biochemical and physi-
cal characteristics of the bioactive and phytochemical compounds in plants and food.
Several methods have been developed through the years, and widely applied to mon-
itor the antioxidant capacity, including: ORAC (oxygen radical absorbance capacity);
TRAP (total radical-reducing antioxidant potential); FRAP (ferric-reducing antioxi-
dant potential); and TEAC (Trolox equivalent antioxidant capacity). The advantages
and disadvantages of these methods have been reviewed (Prior et al., 2005). CAA
(celluare antioxidant activity) and DPPH assays have also been reported and applied
to evaluate the antioxidant capacity of fruits and vegetables (Wolfe et al., 2008). The
ORAC assay has been considered by the U.S. Department of Agriculture (USDA) as
the official method for antioxidant-capacity analysis, and tables of ORAC values for
phytochemicals, foods, and single antioxidants, are available on the USDA website
(http://www. usda.gov/wps/portal/usdahome).
A high positive correlation was found between total phenolic and flavonoid con-
tent, implying that flavonoids constitute an important group of phenolic compounds
in peaches and nectarines (Cantín et al., 2009). Moreover, a linear positive relation-
ship was observed between antioxidant capacity using the DPPH assay, and total
phenolics for the flesh of the peach and nectarine genotypes, and has also been
observed for apricots and plums.
Antioxidant properties of fruits and vegetables mainly contributed to their poly-
phenols and vitamin content. A study performed in our laboratory found significant
differences in flavonoids, such as cyanidin-3-glucoside and pelargonidin-3-glucoside,
272 Advances in Postharvest Fruit and Vegetable Technology

and the antioxidant capacities of strawberry fruit, at different development stages.

No obvious correlation between total phenolic compounds and ORAC and FRAP
were found in strawberries (Li et al., 2013). A study performed on apples and pears
found significant differences in antioxidant capacities in the presence or absence of
the peel. It appears that on the basis of weight, the peel contribution to phenolic con-
tent and the ORAC value is several times higher than that of the pulp.

12.3.1 Genetic Factors Affecting Bioactive

Compounds and Phytochemicals
Production and maintenance of bioactive compounds and phytochemicals in fruits
and vegetables are affected by any pre- and postharvest factors, including agriculture
practices, environmental factors, genetics, harvest maturity, postharvest storage, and
processing. Among them, genetics may be the primary factor.
Significant variation in the level of anthocyanins content between five commercial
strawberry cultivars and three breeding lines were found (Fredericks et al., 2013).
One breeding line (BL 2006-221) was an exceptional source of anthocyanins (~1 g/kg
fresh weight), with approximately double the level of current commercial cultivars.
Hue angle and anthocyanin concentration also showed a good correlation. This indi-
cates that a great divergence of anthocyanin content exists in strawberry fruit.
By combining the metabolomic data (LC–MS), analysis with genetic linkage
maps, 488 mQTLs (metabolite quantitative trait loci) were detected in peel, and
254 mQTLs in apple flesh, using the software MetaNetwork (Khan et  al., 2012).
Despite the fact that half of the metabolites did not have mQTL detected, procyani-
dins, phenolic esters, (+)-catechin, (–)-epicatechin, and kaempferol hexose rhamnose,
showed similar segregation patterns, apparently being controlled by the same domi-
nant and recessive alleles of LG16 from the cross of “Prima” and “Fiesta.” Structural
genes involved in the phenylpropanoid biosynthetic pathway were located using the
apple genome sequence. The structural gene leucoanthocyanidin reductase was in the
mQTL hotspot on LG16, with seven transcription factor genes (Khan et al., 2012).
A single QTL responsible for up to 62% of the variation in the anthocyanin con-
tent was mapped on a Syrah X Grenache F1 pseudo-testcross in grapes (Fournier-
Level et al., 2009). Among the 68 unigenes identified in the grape genome within the
QTL interval, a cluster of four Myb-type genes was selected on the basis of physi-
ological evidence (VvMybA1, VvMybA2, VvMybA3, and VvMybA4). From a core
collection of natural resources, 32 polymorphisms revealed significant association,
and extended linkage disequilibrium was observed. Using a multivariate regression
method, it was demonstrated that five polymorphisms in VvMybA genes, except
VvMybA4, accounted for 84% of the observed variation. All these polymorphisms
led to either structural changes in the MYB proteins, or differences in the VvMybAs
promoters. It was concluded that the continuous variation in anthocyanin content
in grape was explained mainly by a single gene cluster of three VvMybA genes.
Recent studies on 20 tomato cultivars and breeding lines indicated that the levels of
phytochemicals (carotenoids and phenolic compounds), and their antioxidant activ-
ity, were significantly dependent on the genetic background (Li et al., 2011). Similar
Advances in Postharvest Maintenance of Flavor and Phytochemicals 273

results were reported, which showed that the content and activity of total phenolics,
anthocyanins, flavonoids, and vitamin C, were significantly different in genotypes
from peach and nectarine breeding progenies. Recently, an integrated fruit quality
gene map of Prunus, containing 133 genes, putatively involved in the determination
of fruit texture, pigmentation, flavor, and chilling injury resistance, was presented
(Martínez-García et al., 2013).
These results confirm the importance of genotype on the availability of bioactive
compounds and the antioxidant capacity of peach and nectarine fruits and, conse-
quently, on their benefits to health. Therefore, the peach cultivars used as progenitors
in the crosses of a breeding program have a vital importance to release new culti-
vars with high bioactive compounds content. On the other hand, the high number
of evaluated genotypes, from different genetic origins, and with a large phenotypic
variability, constitutes a considerable contribution to peach species, and especially
for breeding purposes.

12.3.2 Postharvest Storage Effects on Bioactive

Compounds and Phytochemicals
Changes in both the quality and phytochemical composition of plants can occur
rapidly, depending on postharvest handling, such as storage and processing con-
ditions. Such changes may not always result in reduction of the health-promoting
compounds. The concentration of phytochemicals and antioxidant activity in some
fruits and vegetables were actually enhanced by postharvest storage and processing
parameters (Bengtsson, 2010). The two major chemical changes causing deteriora-
tion are lipid oxidation, and nonenzymatic browning, during storage and food pro-
cessing, which can lead to altered color and flavor. Lipid oxidation is influenced by
light, oxygen, temperature, and water activity, and the presence of catalysts, such as
transition metals iron and copper. Nonenzymatic browning can occur easily during
the storage of dried and concentrated foods. Different phytochemicals are affected
by these factors differently. Carotenoids are very sensitive to heat, and can incur
significant losses during different vegetable processing steps (Tiwari and Cummins,
2013). The main cause of carotenoid degradation in foods is oxidation.
Significant differences in anthocyanin concentration, as a result of fruit-ripening,
was shown in two strawberry cultivars (“Mira” and “Honeoye”), when expressed as
total anthocyanins content of total 520 nm area count, equivalent to either cyanid-
ing 3-O-glucoside or pelargodin-3-glucoside. Anthocyanin content was significantly
higher in red fruit of both cultivars, being 22.9 and 17.07 mg/100 g FW of Cy-3-glc
equivalents, or 58.93 and 43.9 mg/100 g FW Pel-3-glc, respectively. In red ripe fruit,
“Honeoye” showed higher anthocyanin content than “Mira.” Meanwhile, a signifi-
cant decrease in total flavonoid compounds during ripening was observed, while
no significant decrease in total phenolic compounds was found (Li et  al., 2013).
A ­quadratic relationship in the antioxidant capacity, measured as ORAC, was found
in “Mira” at three ripeness stages, while a significant decrease of 15% of the anti-
oxidant capacity, measured as FRAP, was found in both cultivars, with advanced
ripeness at the red—as compared with the white—stage.
274 Advances in Postharvest Fruit and Vegetable Technology

Flavonoids and other phenolic compounds are relatively stable at high tempera-
ture, and over long storage. Phenolics in plants exist in both free and conjugated
forms. Postharvest loss of phenolics is mainly due to enzymatic oxidation by poly-
phenol oxidase and peroxidases (Wang et al., 2007). Degradation of anthocyanins
is pH dependent. Percentage degradation of total anthocyanins, and total antioxi-
dant activity in unblanched blueberry juice (conventional and organic), after five
months of storage at 23°C, was reported to be 72%–79% and 21%–43%, respectively
(Syamaladevi et al., 2012). MCP treatment was found to maintain the ascorbic acid,
carotenoid, total phenolic, and flavonoid content of mango fruit (Sivakumar et al.,
2011). Apples treated with 1-MCP had no significant change in overall phenolic
content, and antioxidant activity, although the effects on individual phenolic com-
pounds, and on polyphenols, in different parts, varied. Similar results were found
in cherry fruit, where cherries treated with 1-MCP, hexanal, or a combination of
both, showed enhanced fruit quality and extended shelf-life, but no significant effect
on polyphenols, such as anthocyanins and phenolic acids (Sharma et al., 2010). As
briefly discussed above, although the effect of postharvest storage of fresh produce
on phytochemicals is multifaceted, optimizing the various parameters can lead to
good retention of nutritionally important food bioactives, such as those with strong
antioxidant activities. Studies have also shown that the phytochemical content of a
particular cultivar can vary significantly, due to other factors.
Controlled atmosphere (CA) and MAP have been widely applied on many fresh
fruits and vegetables, as they are very effective in maintaining the quality, and
extending the marketability of fresh produce. Many studies have also shown that
CA or MAP technologies offer the possibility to retard the respiration rate, maintain
bioactive compounds, and extend the shelf-life of fruits and vegetables, as compared
with conventionally stored or packaged samples. Overall, total phenolic, flavonoid,
and anthocyanin concentrations, as well as antioxidant activity, were relatively stable
during air and CA storage. In air-stored fruit, total phenolic concentrations were
higher in the peel of 1-MCP treated fruit, than in the control fruit, but slightly lower
in the flesh of 1-MCP-treated fruit. In CA-stored fruit, interactions between O2 par-
tial pressures, temperature, and storage duration, were detected, but overall, few
consistent trends were observed. However, flavonoid concentrations were higher in
the flesh of 1-MCP-treated than untreated fruit kept in 2 kPa O2 while anthocyanin
concentrations, only measured in the peel, were not affected by 1-MCP treatment.
There were no correlations found between total phenolics and antioxidant activity.
Ascorbic acid concentrations declined in both peel and flesh tissues of untreated and
1-MCP-treated fruit stored in air, while changes of ascorbic acid concentrations in
CA-stored fruit were inconsistent (Fawbush et al., 2009). For better phytochemical
retention, shelf-life and phytochemicals of broccoli florets seemed to be maintained
with polypropylene microperforated film and refrigerated conditions (Nath et  al.,
2011). Several families of phytochemicals, such as phenolic acids, isoflavones, fla-
vones, flavonols, and glucosinolates, were determined in both fresh and fresh-cut
samples, including tomato, carrot, grape, eggplant, and broccoli (Alarcón-Flores
et  al., 2014). Both samples of produce have potential and similar beneficial prop-
erties, regarding their content of phytochemicals, except tomato, which should be
consumed as fresh. Processes such as slicing, grating, and dicing, as well as storing
Advances in Postharvest Maintenance of Flavor and Phytochemicals 275

conditions (temperature and light), were observed to impact the content in eggplant;
the content of phenolic acids is statistically different, depending on the presentation.
The content of phytochemicals was higher when fresh-cut carrots were stored at 4°C,
regardless of the presence or absence of light.

12.4  FUTURE RESEARCH PERSPECTIVES

From the postharvest perspective, research plays a critical role in both the frontiers
of flavor and phytochemicals. Further research is needed on understanding how the
agriculture production system affects flavor and phytochemicals, and on aligning
research between nutrition and agricultural production. Research efforts on quality
evaluation need to shift from yield and quantity to eating-quality factors (such as
taste, flavor, and nutritional value), which have positive effects on fruit and vegetable
consumption. More research and evidence is also needed on the content and bio-
availability of bioactive compounds.
Production of flavor compounds and bioactive compounds is the result of com-
plex metabolic networks involving many pathways and control mechanisms. It is
necessary to remember that volatile biosynthesis pathways and bioactive compounds
are only a part of the complex network of fruit metabolism during ripening, and is
influenced by many factors, such as genetics, production practices, and postharvest
handling. The metabolomic diversity is also caused by low enzyme specificity, and
is directly related to the availability of substrates. Future work must, therefore, take
into account the entire metabolic pathway, rather than a single enzyme or section
of the pathway. Flavor/aroma is one of the most important quality indices of fruits
and vegetables. It contributes not only to the attractiveness of food, but may also
be associated with nutritional quality, due to many commonly shared precursors in
secondary metabolism (Klee, 2010). This implies that new research needs to address
the whole network of biology, rather than individual—or a group of—metabolites.
This system approach employs state-of-the-art metabolomics, genomic, and pro-
teomic tools, to study fundamental metabolism, and its regulation and localization.
Combining results of genetic, chemical and sensory properties, will lead to a better
understanding of how to optimize and retain fruit flavor quality in the marketplace,
for the benefit of both consumers and the fruit industry.
Volatile biosynthesis in both climacteric and nonclimacteric fruit is highly inte-
grated with fruit-ripening and senescence. Therefore, a better understanding of fruit-
ripening and its triggers will help in our understanding of fruit volatile production
(Alexander and Grierson, 2002). Linkages of flavor biosynthesis with climacteric
respiration, endogenous ethylene content, ethylene biosynthesis, and its response to
the inhibitors AVG and 1-MCP, will provide new insights into control mechanisms.
Using AAT, LOX, ADH, PDC, and other enzymes, as examples, it has been dem-
onstrated how these enzymes influence fruit volatile biosynthesis in fruit. While
precursor studies indicate that AAT is not the limiting factor in volatile production,
it may also imply that both fatty acids and branched amino acid substrates were
physically unavailable to the enzyme. Due to overwhelming evidence that substrates
may be the bottleneck of volatile production in most fruit, it becomes very important
to clarify the compartmentalization of volatile biosynthesis within the fruit cell, in
276 Advances in Postharvest Fruit and Vegetable Technology

order to understand the biosynthesis of those substrates or precursors, and their com-
partmentalization and transport in the cell. Information on aroma threshold levels
sheds light on the nature of chemicals that may contribute to human perception of
aroma volatiles.
While precursor studies of early harvested or 1-MCP-treated fruit indicate that
AAT is not the limiting factor of volatile production, it may also imply that the sub-
strates were physically unavailable to the enzymes. Understanding where substrates
are produced, and where enzymes are localized, will improve our understanding of
the volatile biosynthesis system. Immunolocalization, immunoblot, and fluorescence
imaging techniques, could help to determine protein expression, localization, activ-
ity state, and cell compartmentation (Giepmans et al., 2006). Information on aroma
thresholds will continue to aid in the identification of compounds that contribute to
human perception of fruit flavor. Little information is available about the sensory
contribution of many volatiles. Of particular interest is an objective olfactory descrip-
tion of fruit “freshness,” “ripeness,” “off-flavor” and “over-ripeness.” Combining
sensory and instrumental analysis should refine our understanding of fruit-ripening
and consumer preferences, and help to develop production and postharvest handling
technologies to optimize fruit flavor quality (Song and Forney, 2008).
Similar to flavor, bioactive compounds and phytochemicals in fruits and vegeta-
bles can provide health benefits beyond their basic nutritional values. Phytochemicals
such as carotenoids, phenolics, and glucosinolates are among the most important
food bioactives that have positive impact on human health. In addition to the unique
phytochemical profile of different plants, the amount and composition of a particular
plant or food can also be changed by the various conditions during growth, posthar-
vest storage, and processing. Fully understanding the roles of these factors will help
the development of strategies to preserve the bioactive components, and maximize
their bioavailability, and ultimately, their potential health benefit. This is an under-
taking that requires a multidisciplinary approach, and support from the agricultural
and agrifood sectors.
Genetic markers can assist fruit breeders to assess genetic diversity of the germ-
plasm, determine heritability, and predict cultivars. A major advantage of using
markers in fruit breeding is the improved efficiency of enabling early selection for
any quality traits, simultaneous selection for multiple traits, including resistance
gene pyramiding, and selection for traits that are expensive to screen by phenotype.
According to published data, markers have been increasingly used for selection in
apple breeding. To increase selection efficiency, and to reduce MAS cost in apple
breeding, knowledge of the most useful phenotypic characters is essential. The latest
advances in apple genetics offer unprecedented opportunities for cultivar improve-
ments. Future apple breeding programs should consider the creation of new cultivars
combining fruit quality features such as texture, storability, well-balanced acid/sugar
ratio, and desirable aroma (Ulrich and Dunemann, 2012).
Improvement of the flavor and phytochemistry of fruits and vegetables will only
be successful through multidisciplinary research. Metabolomics, genomic, pro-
teomics, and integrative multiomics techniques, will provide the essential informa-
tion and knowledge on the production and regulation of flavor and phytochemical
compounds in fruits and vegetables, but also reveal biological impact on animal and
Advances in Postharvest Maintenance of Flavor and Phytochemicals 277

human nutrition. Several components of fruits and vegetables have been isolated,
structures elucidated and tested, singularly or in mixtures, to determine their effect
on cells and animals.
Metabolomic analysis utilize GC, LC-MS, LC-MS (lipid), and nuclear mag-
netic resonance (NMR) spectroscopy, for structure elucidation and multivariate, a
statistical analysis to identify important relationship among biological conditions.
Metabolomics is particularly important in the plant field, because plants produce a
huge diversity of metabolites—far more than are produced by animals and micro-
organisms (Saito and Matsuda, 2010). The manipulation of plants and the search
for new plants endowed with high antioxidant potential are conducted in tandem
with metabolomic analysis, which is integrated with nutrigenetics and nutrigenom-
ics. These disciplines, which have evolved rapidly in recent years, have increased
our knowledge of the interactions between life processes and specific components
of our diet. The co-occurrence principle of transcripts and metabolites, particularly
transcriptome coexpression network analysis, is powerful for decoding functions of
genes, not only in a model plant such as Arabidopsis but also in crops and medici-
nal plants. mQTL analysis, along with scoring of gene expression and agronomical
traits, is beneficial for crop-breeding. Although comprehensive coverage of metabo-
lomic analysis is achieved not by a single analytical technology but by multiparallel
complementary technologies, it increases the challenge level of the annotation rate
of unknown signals.
Proteomics is the study of “the entire protein complement expressed by a genome
in a cell or tissue type,” and is a major research tool in the postgenomics era (Pandey
and Mann, 2004). It provides an essential link between the transcriptome and metab-
olome, and is becoming an important research platform, along with genomics, as the
latter is greatly enhanced through identification of corresponding specific proteins
derived from transcripts. The development of proteomics originated from “genom-
ics”; however, it has developed to cover all aspects of protein research in a cell, not
only the abundance and changes in time and in association with biological behaviors,
but also post-translational modifications (PTMs) related to the biological functions,
and protein–protein interactions. Recent and continuing development in proteomic
technologies has demonstrated that proteomics is the crucial element of the “omics”
approach, and contributes greatly to a new understanding of biological systems,
especially for fruit and vegetables (Palma et al., 2011). Systems biology, data-driven
by metabolomics and other “omics,” will play a key role in understanding plant sys-
tems, and developing further biotechnology applications. As the proteome consists
of all proteins present in a specific cell type, it may actually provide a better indica-
tion of the biological effect of phytochemicals in human nutrition studies.
Combining gene and protein expression profiling in colonic cancer cells, Herzog
et al. (2004) identified the flavonoid flavone, present in a variety of fruits and veg-
etables, as a potent apoptosis inducer in human cancer cells (Herzog et al., 2004).
Flavone displayed a broad spectrum of effects on gene and protein expression that
related to apoptosis induction and cellular metabolism. The effect(s) of the flavo-
noid quercetin on normal and malignant prostate cells was evaluated, and possible
target(s) of quercetin action was identified. This finding demonstrated that querce-
tin treatment of prostate cancer cells resulted in decreased cell proliferation and
278 Advances in Postharvest Fruit and Vegetable Technology

viability. Quercetin promoted cancer cell apoptosis by downregulating the levels of

heat shock protein 90 (Aalinkeel et al., 2008).
These studies demonstrate that, indeed, proteomics analysis, particularly when
combined with transcriptome analysis, may reveal effects of dietary phytochemicals
relevant in cancer prevention. Metabolomics approaches aim to identify changes
in relevant physiological processes, based on modifications in the occurrence and
concentrations of all end-products of metabolic enzyme activity, in response to
exposures or changes in environmental conditions. One of the biggest advantages
of this technique is that it can be applied to biological samples like urine, serum,
or plasma, making it very suitable for biomonitoring purposes. Although metabo-
lomics analysis can bring comprehensive understanding of biological processes a
step forward, studies on the effects of dietary phytochemicals are still very sparse.
For instance, although each “omics” technique has its own merits and limitations,
combined analysis and interpretation of multiomics data are likely to offer the best
opportunities for comprehensive understanding of the biological influences induced
by phytochemicals. Data integration of different “omics” techniques will become
increasingly important in any system biology study, including postharvest and
human nutrition.

ACKNOWLEDGMENT
The author thanks Dr. C. Forney at AAFC for his critical review of this manuscript
and his constructive suggestions.

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 13 Metabolomics Tools for
Postharvest Quality and
Safety of Fresh Produce
Sukhvinder Pal Singh

CONTENTS
13.1 Introduction................................................................................................... 285
13.2 Metabolomics: Analytical Approaches.........................................................286
13.3 Metabolomics Applications in Fresh-Fruit Quality....................................... 287
13.3.1 Breeding for Postharvest Quality...................................................... 288
13.3.2 Fruit-Ripening................................................................................... 289
13.3.3 Physiological Disorders..................................................................... 291
13.3.3.1 Superficial Scald in Apples................................................. 291
13.3.3.2 Flesh-Browning in Apples.................................................. 292
13.3.3.3 Internal Browning in Pears................................................. 293
13.3.3.4 Puffing Disorder in Citrus.................................................. 293
13.3.4 Preharvest Treatments....................................................................... 294
13.3.5 Postharvest Treatments...................................................................... 295
13.3.6 Postharvest Storage Conditions......................................................... 296
13.3.7 Pre- and Postharvest Diseases........................................................... 297
13.4 Metabolomics Applications in Safety of Fresh Produce............................... 298
13.4.1 Detection and Quantification of Chemical Residues......................... 298
13.4.2 Detection of Mycotoxins...................................................................300
13.4.3 Detection of Pathogens......................................................................302
13.4.4 Traceability, Fraud, and Malpractices............................................... 303
13.5 Conclusions....................................................................................................304
References...............................................................................................................304

13.1 INTRODUCTION
Metabolomics is a novel experimental methodology categorized as an “omics”
approach, along with genomics, transcriptomics, and proteomics (Hertog et  al.,
2011), and is defined as the comprehensive, simultaneous determination of endog-
enous metabolites at the molecular level and their global and dynamic changes
over time in complex multicellular systems as a consequence of biological stimuli
or genetic manipulation or both (Hu and Xu, 2013). Metabolomics is often used
in combination with the other “omics” approaches for a deeper understanding of

285
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biological processes, especially metabolism, through global studies on metabolites

and their concentration and dynamics within complex samples. As metabolites are
considered the downstream products of cellular regulatory processes, metabolomics
data can precisely characterize cells, tissues, or whole organisms by defining specific
biochemical phenotypes that are representative of physiological or developmental
states. The metabolome is the holistic quantitative set of low-molecular-weight com-
pounds (<1000 Da), including many hundreds or thousands of molecules such as
carbohydrates, vitamins, lipids and amino or fatty acids. The number of metabolites
in the plant kingdom is considered to be far greater than that in the animal kingdom,
and is estimated to exceed 200,000 (Fiehn, 2002). This large number of metabo-
lites is due to the great diversity of metabolic pathways that each plant species has
evolved, to survive under varying environmental conditions.

13.2  METABOLOMICS: ANALYTICAL APPROACHES

The complexity and diversity of metabolites in terms of their number, localiza-
tion, and chemical nature, pose considerable challenges in their analysis, and data
interpretations. Therefore, the metabolome analysis is broadly classified into two
approaches: targeted and untargeted. Targeted metabolomic analysis involves identi-
fication and absolute quantification of a limited number of metabolites under a given
set of conditions, while an untargeted approach is aimed at qualitative analysis (iden-
tification and relative quantitation) of the maximum possible metabolites in a system
(cell, tissue or organ). The targeted metabolomics typically represents a bottom-up
approach compared to the top-to-bottom approach in untargeted metabolomics. Both
of these approaches require different types of analytical platforms and bioinformat-
ics tools. However, the enhanced capabilities of new analytical systems have enabled
the broadening of scope, and increased sensitivity and resolution for targeted as well
as untargeted analyses.
There are several analytical options available to find an answer to metabolic
inquiry. A typical analytical system will consist of a separation step, followed by
detection, and then data processing. Three separation techniques—gas chromatogra-
phy (GC), liquid chromatography (LC) and capillary electrophoresis (CE)—are most
commonly employed in metabolomics studies. The choice of a separation technique
depends on the chemical nature and sensitivity of small molecules, and compat-
ibility with the detection system. The separation system is hyphenated to a detection
system. The approach based on mass spectrometry (MS) has recently emerged as
the technique of choice, considering its advantages over nuclear magnetic resonance
(NMR) systems. MS offers distinct advantages of unparalleled sensitivity and speci-
ficity, high resolution and wide dynamic range, enabling comprehensive quantita-
tive and qualitative measurement of large-scale metabolites in complex biological
samples. On the other hand, NMR is also used for metabolomics studies because
of its simplicity, rapidity, high selectivity and nondestructive nature, but relatively
lower sensitivity.
Gas chromatography–mass spectrometry (GC–MS) has been regarded as the
gold standard for metabolomics as the platform that provides reproducible reten-
tion characteristics, high chromatographic resolution, high sensitivity, reproducible
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 287

production of mass spectra, which aids metabolite identification, and the avail-
ability of MS libraries, which are much less common for liquid chromatography–
mass spectrometry (LC–MS) (Lisec et al., 2006). Coupling LC to MS enables the
detection of multiple metabolite classes in a single analysis, even in a very com-
plex matrix such as rich plant extracts. The technology for liquid-phase separation
has also advanced in recent years, with the introduction of ultra-performance LC
(UPLC) that enables faster separation with better separation and sensitivity as com-
pared to high-performance LC (HPLC). The analytical instruments typically used
in metabolomics experiments differ in their ionization technology, for example,
electron ionization (EI), chemical ionization (CI), electrospray ionization (ESI),
atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorp-
tion/ionization (MALDI), and the type of mass analyzer, for example, quadrupole,
triple-quadrupole (QQQ), ion-trap, time-of-flight (TOF), and Fourier-transform ion
cyclotron resonance (FT-ICR) mass analyzer. Samples can be directly analyzed by
direct infusion technique in MS or resolved primarily by different chromatographic
techniques. The EI and ESI sources of ionization have been most commonly used
in the GC- and LC-based metabolomics approaches, respectively. Despite the recent
extensive development in analytical methods, the ultimate goal of plant metabolo-
mics—to gain a complete overview of the metabolite complement of a plant in one or
a small series of analyses—is currently impossible. However, continued technologi-
cal improvements in the applications of hybrid technologies and combinations will
help develop a better understanding of the metabolome. The detailed discussion on
analytical methods in metabolomics is beyond the scope of this chapter.
In addition, these analytical platforms generate large data-sets, which require
further complex processing to derive meaningful interpretations. The analytical
systems supplied by various companies often offer informatic solutions to process
the data-sets for various functions such as peak-filtering, alignment, data-reduction,
and multivariate statistical analysis (e.g., MarkerLynx from Waters, MarkerView
from AB Sciex and MassProfiler from Agilent). In addition, there are several public-
domain databases (Metlin, MassBank, XCMS, MZmine etc.) that are available to
conduct data-processing and identification of metabolites based on mass spectra and
other features such as exact mass, elemental composition, etc. The correct identifica-
tion of a metabolite with high level of confidence is still a major challenge in global
metabolomics studies, which tend to separate and detect a huge range of metabolites
simultaneously.

13.3  METABOLOMICS APPLICATIONS IN FRESH-FRUIT QUALITY

Among different “omics” tools, metabolomics is more relevant to postharvest
research, unless used in tandem with others in a systems-biology approach. In com-
parison with genome, transcriptome and proteome, metabolome can be best studied
in response to the environmental factors. The epigenetic, posttranscriptional, and
posttranslational modifications often limit the scope and robustness of genomics,
transcriptomics and proteomics, respectively. Postharvest science involves studying
the effects of various external environmental stimuli (such as temperature, storage
atmosphere, heat and cold treatments, etc.) on the tolerance and quality of products.
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These stimuli directly or indirectly influence the metabolome expressions, which

are manifested in different forms in terms of quality and the postharvest life of
a commodity. The metabolome of fresh fruits and vegetables not only represents
small molecules as intermediary and final products of various metabolic pathways,
but these molecules have key roles in determining quality attributes such as color,
taste, aroma and nutrition. Postharvest factors, such as treatment, storage, and dis-
tribution, act as external stimuli having both positive and negative consequences on
quality and safety of fruit and vegetables (Hertog et al., 2011). These can be exam-
ined using metabolomic evaluation techniques; however, to date, research employing
untargeted metabolic profiling to study postharvest issues, is relatively uncommon.

13.3.1 Breeding for Postharvest Quality

In clinical chemistry, metabolomics is applied in the discovery of biomarkers for
diagnosing disease, prognosis, and risk-reduction. However, the expansion of this
approach in plant sciences is rapidly discovering new biomarkers for the determina-
tion of heredity of biochemical traits, linking traits to specific genes, and screen-
ing germplasm for bioactive compounds of interest (Zanor et al., 2009). Untargeted
metabolic fingerprinting techniques have been used to distinguish individuals with
unique quality characteristics within breeding and wild populations. While targeted
metabolite analysis of mapping populations is useful for limited discovery of quality-
related metabolic quantitative trait loci (QTL), untargeted biochemical phenotyping
of genetically mapped populations can reveal multiple metabolic QTLs that com-
prise sensory traits (Wahyuni et al., 2013). Recently, a genome-wide metabolomic
resource for tomato fruit from Solanum pennellii has been developed (Perez-Fons
et al., 2014). A multiplatform metabolomic analysis, using NMR, MS, and HPLC, of
introgression lines of Solanum pennellii with a domesticated line, was conducted to
analyze and quantify alleles responsible for metabolic traits. QTL for health-related
antioxidant carotenoids and tocopherols, as well as molecular signatures for some
2000 compounds, have been reported (Perez-Fons et al., 2014). Correlation analy-
ses have also revealed intricate interactions in isoprenoid formation in the plastid
that can be extrapolated to other crop plants. Identification of metabolite alleles in
introgression lines, as well as computational integration of the data-sets, has been
carried out to construct metabolic networks for the tomato fruit metabolome and
the individual pathway components. These data-sets represent a valuable and timely
resource to fully capitalize on the sequencing of tomato genomes.
Metabolomics approaches have also been successfully used to complement asso-
ciation genetics studies to identify QTL for important quality traits in apple fruit.
The LC–MS analysis of fruit extracts from a segregating F1 population of a cross
between apple cultivars “Prima” and “Fiesta” has been used to integrate metabolite
data with the existing reference map for genetic linkage, thus allowing the mapping
of metabolite quantitative trait loci (Maliepaard et al., 1998; Khan et al., 2012). The
same approach was used successfully to map metabolic QTL (mQTL) for apple fruit
volatiles (Dunemann et al., 2009). Apple linkage group 16 was found to contain an
mQTL hotspot for various phenylpropanoid metabolites, which correlated with the
location of genes putatively involved in the phenylpropanoid pathway (Khan et al.,
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 289

2012). This knowledge can now be used in marker-assisted breeding, which should
be greatly aided by the available apple genome sequence. Cuthbertson et al. (2012)
employed a GC–MS approach to evaluate the use of metabolite patterns to differ-
entiate fruit from six commercially grown apple cultivars in central Washington
State in the United States. The principal component analysis (PCA) of apple fruit
peel and flesh metabolome data indicated that individual cultivar replicates clustered
together and were separated from all other cultivar samples and this observation
was further supported by a hierarchical clustering analysis. An evaluation of PCA
component loadings revealed specific metabolite classes that contributed the most to
each principal component, whereas a correlation analysis demonstrated that specific
metabolites correlated directly with quality traits such as antioxidant activity, total
phenolics, and total anthocyanins, which are important selection parameters in some
breeding programs.

13.3.2 Fruit-Ripening
Metabolic analysis has been an integral part of postharvest science for over a c­ entury.
However, the targeted metabolites quantified for quality analysis were limited in
number. The new developments in analytical platforms and informatics tools have
empowered postharvest researchers to gain better insights into fruit-ripening and to
integrate the data from all “omics” for a systems-biology approach. To date, most of
the metabolomics studies have focused on changes in metabolome during the growth,
maturation and ripening stages in the tomato fruit (Carrari et al., 2006; Moco et al.,
2006; Osorio et al., 2012), strawberry (Zhang et al., 2011), peach (Lombardo et al.,
2011) grapes (Toffali et  al., 2011; Zamboni et  al., 2010), and avocado (Pedreschi
et al., 2014).
The fruit-development, -ripening and senescence processes are important deter-
minants for quality and postharvest storage and shelf-life. Therefore, understanding
the metabolic events involved in these processes is critical to make harvest deci-
sions, choosing the appropriate postharvest handling and storage procedures and
managing product quality for the consumer. The integration of “omics,” including
metabolomics, provides more information about the regulatory networks involved
in controlling vital processes, such as fruit-ripening. Osorio et al. (2012) followed
a systems-biology approach to reveal a network of candidate regulators that play
important roles in tomato fruit-ripening. The combination of transcriptome, pro-
teome, and targeted metabolite analysis helped to refine the ethylene-regulated tran-
scriptome of tomato fruit and added to the knowledge of the role of ethylene in both
protein and metabolite regulation in tomato-ripening. The metabolite abundance of
specific compound classes, such as tricarboxylic acid (TCA) cycle organic acids and
cell-wall-related metabolites, appears to be strictly controlled, with specific com-
pounds influenced by ethylene, transcriptional control, or both. This systems-biology
approach helped identify areas of metabolism that seem to be of high importance to
the ripening process, such as hormones and cell-wall metabolism in ethylene per-
ception. Therefore, the integrated analysis has enabled the discovery of additional
information for the comprehensive understanding of biological events relevant to
metabolic regulation during tomato fruit development. The outcomes of this work
290 Advances in Postharvest Fruit and Vegetable Technology

will provide potential targets for the engineering of metabolism to facilitate the con-
trolled modulation of ripening in tomato fruit (Osorio et al., 2012).
In peach fruit, Lombardo et al. (2011) showed that the principal role of amino acid
metabolism during the early stages of ripening were as substrates for the phenylpro-
panoid pathway. These results suggest that enzymes involved in amino acid metabo-
lism may be useful biomarkers for the different developmental stages as well as key
to determining the final quality of peach fruit. Other metabolic determinants for
sucrose levels, invertase activity for early stages of ripening, and sucrose cycling for
ripening, were also inferred by Lombardo et al. (2011). They also highlighted the rel-
evance of stone-formation by lignification of the fruit endocarp layer, which affects
primary metabolism to fulfill this process. Finally, the relevance of the posttran-
scriptional regulation of enzyme activity indicated the need for integrated studies
of metabolic pathways, combining analyses of metabolites, transcripts, and proteins
(Lombardo et al., 2011).
Seven developmental stages of strawberry fruit—to maturation and ripening—
were subjected to metabolic profiling of both polar and nonpolar compounds to
monitor the alterations in several major groups of compounds (Zhang et al., 2011).
The results of metabolic profiling, conducted using GC–MS and HPLC–PDA sys-
tems, were consistent with strawberry pigment- and flavor-formation, using mostly
materials from primary metabolism. Each stage of fruit development represented
its unique metabolic profile, with the most drastic changes occurring at the transi-
tion toward the red-ripened stage. Like peaches, amino acid biosynthesis was found
to play an important role in generating several classes of compounds related to the
quality of the strawberry fruits (Zhang et al., 2011). This information has potential to
be translated by the strawberry breeders to detect and monitor key components that
are important for these output traits.
The comparison of metabolic profiling during the development of peach
(Lombardo et al., 2011), tomato (Osorio et al., 2012), strawberry (Zhang et al., 2011),
and grapes (Toffali et al., 2011; Zamboni et al., 2010) indicates that each fruit f­ ollows
a diverse metabolic program. Furthermore, these fruits are models for studies of
ripening behavior where tomato and peach are defined as climacteric fruits, while
strawberry and grapes are considered nonclimacteric. Since great differences in
terms of morphology, physiology, and biochemistry are found among fruits, it seems
reasonable that singular metabolic programs support the differential developmental
processes. A recent in silico study on the comparative metabolomes of three climac-
teric (peach and two tomato cultivars) and two nonclimacteric species (strawberry
and pepper) was conducted by Klie et al. (2014) with data on the metabolic profiles
of these fruits during development and ripening stages. The analysis was based on
an extension to principal component analysis, called STATIS, in combination with
pathway over-enrichment analysis. STATIS is a novel tool that can be used to identify
the metabolic processes that are affected to similar extent during fruit-development
and -ripening. These results ultimately provide insights into the pathways that are
essential during fruit-development and -ripening across species (Klie et al., 2014).
This study is an example of the application of new informatic tools to metabolomics
data, to derive an overarching view of the biological processes involved in matura-
tion and ripening.
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 291

The application of the metabolomics tool has also been extended to determine the
sensorial quality of some apple cultivars (“Golden Delicious,” “Liberty,” “Santana,”
and “Topaz”) grown in organic (ORG) versus integrated production (IP) systems
(Vanzo et al., 2013). Metabolic profiling revealed that significantly higher total phe-
nolic content in ORG fruit was found in “Golden Delicious,” whereas differences
in other cultivars were not significant. Targeted profiling of multiple classes of phe-
nolics confirmed the impact of the production system on the “Golden Delicious”
phenolic profile, with higher levels of 4-hydroxybenzoic acid, neo- and chlorogenic
acids, phloridzin, procyanidin B2 + B4, quercetin-3-O-glucoside and quercetin-3-O-
galactoside, kaempferol-3-O-rutinoside, and rutin being found in ORG fruit. The
results also suggested that scab resistance in some apple cultivars is influenced by
the phenolic biosynthesis in relation to the agricultural production system (Vanzo
et al., 2013). Although such results are not conclusive enough to define the impact
of production systems on metabolic profiles of fruit, similar studies have significant
scope for future research to discover biomarkers for organic (and other) produce.
Postharvest ripening behavior of “Hass” avocado is heterogeneous and complex.
A fruit biopsy methodology was followed to derive samples of mesocarp tissue,
which were subjected to untargeted and targeted metabolomics (Pedreschi et  al.,
2014). The results showed that while C7 sugars (mannoheptulose and perseitol), dry
matter, and total Ca2+ were not correlated with time to reach edible ripeness, untar-
geted metabolomics profiling of polar and semi-polar compounds (based on GC–MS
and LC–MS platforms), revealed several metabolites, mainly amino acids, that were
related to ripening heterogeneity. The analysis of fatty acids revealed linoleic acid to
be accumulating differentially. In general, the slowest ripening avocados had lower
amounts of precursors of metabolites involved in key metabolic pathways (Pedreschi
et  al., 2014). This study indicated that comprehensive metabolomics may provide
new markers for the avocado-ripening stage at harvest, and will provide greater
insights into the complex ripening physiology of this fruit.

13.3.3 Physiological Disorders
Metabolomics tools are becoming increasing popular to link the development of
postharvest physiological disorders in fruits with some metabolites (Lee et al., 2012;
Leisso et al., 2013; Rudell et al., 2009; Rudell and Mattheis, 2009). This section will
outline the role of metabolomics to uncover the causes of the physiological disorders:
superficial scald in apples, flesh-browning in apples, internal-browning in pears, and
puffing disorder in citrus.

13.3.3.1  Superficial Scald in Apples

Superficial scald in apple has been well studied due to its commercial importance
for the industry. The development of scald has been largely attributed to the oxi-
dation products of α-farnesene and antioxidant metabolism. The application of
metabolomics tools in understanding the chemistry of apple peel in relation to scald
has provided great insights into the metabolic pathways, leading towards develop-
ment of predictive biomarkers for scald. Rudell et al. (2009) employed untargeted
metabolic profiling to characterize metabolomic changes associated with superficial
292 Advances in Postharvest Fruit and Vegetable Technology

scald development in “Granny Smith” apple, following 1-MCP or DPA treatment.

Partial least squares models revealed metabolomic differentiation between untreated
controls and fruit treated with DPA or 1-MCP within one week following initiation
of storage. Metabolic divergence between controls and DPA-treated fruit after four
weeks of storage, preceded scald symptom-development by two months. The study
demonstrated that extensive metabolomic changes in levels of various classes of tri-
terpenoids, including β-sitosterol associated with scald, precede actual symptom-
development (Rudell et al., 2009).
To elaborate the role of phytosterol metabolism in the development of apple scald,
Rudell et al. (2011) studied changes in peel-tissue levels of conjugates of β-sitosterol
and campesterol, including acylated steryl glycosides (ASG), steryl glycosides (SG)
and steryl esters (SE), as well as free sterols (FS) during the period of scald-develop-
ment in response to antiscald postharvest treatments; diphenylamine (DPA), 1-MCP,
and intermittent warming. ASG levels increased and SE levels decreased in untreated
control fruit during storage. Removing fruit from cold storage to ambient temperature
induced rapid shifts in ASG and SE fatty acyl moieties from unsaturated to saturated.
FS and SG levels remained relatively stable during storage but SG levels increased,
following a temperature increase after storage. ASG, SE, and SG levels did not
increase during six months’ cold storage in fruit subjected to intermittent warming
treatment. These results clearly showed that apple-peel phytosterol conjugate metabo-
lism is influenced by storage duration, oxidative stress, ethylene action/ripening, and
storage temperature (Rudell et al., 2011). Leisso et al. (2013) recently showed that the
metabolic profile of control and DPA-treated fruit was divergent after 30 days of cold
storage due to differing levels of α-farnesene oxidation products, methyl esters, phy-
tosterols, and other compounds potentially associated with chloroplast integrity and
oxidative stress response. Leisso et al. (2013) showed evidence of coregulation within
the volatile synthesis pathway, including control of the availability of methyl, propyl,
ethyl, acetyl, and butyl alcohol and/or acid moieties for ester biosynthesis.
The researchers at the USDA (United States Department of Agriculture) Tree Fruit
Research Laboratory in Washington state, United States, have applied metabolomics
tools to discover and validate various risk assessment biomarkers for scald (Leisso
et al., 2013; Rudell et al., 2008, 2009, 2011; Rudell and Mattheis, 2009). These bio-
markers may be useful as apple storage management tools by indicating when and
which fruit are at higher risk to develop scald. By selecting biomarkers for scald using
storage conditions typically required for scald to develop, and then validation under
storage conditions similar to commercial practice, risk assessment tools are expected
to be widely applicable. However, the outcomes of these studies require further valida-
tion under commercial conditions in order to develop a diagnostic tool to accurately,
and in timely fashion, predict the onset and development of superficial scald in apple.

13.3.3.2  Flesh-Browning in Apples

Flesh-browning is a complex physiological disorder that develops in apple during
long-term cold storage and is controlled or aggravated by certain postharvest treat-
ments. A GC–MS-based global metabolomics study has been reported to underpin
the reasons why “Empire” apple fruit were susceptible to flesh-browning at 3.3°C
if treated with 1-MCP (Lee et al., 2012). Most carbohydrates and organic acids in
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 293

flesh tissue were not appreciably affected, but the levels of amino acids and volatile
metabolites were significantly affected by 1-MCP treatment. In addition, sorbitol
and levels of some amino acids were elevated towards the end of storage in 1-MCP-
treated fruit. CA storage resulted in lower levels of many volatile components while
1-MCP treatment reduced these levels further. Lee et al. (2012) further showed that
multiple metabolites were associated with the development of flesh-browning symp-
toms. Unlike other volatile compounds, methanol levels gradually increased with
storage-duration, regardless of 1-MCP treatment, while 1-MCP decreased ethanol
production. The metabolic shifts in response to CA storage and 1-MCP need fur-
ther investigations to understand the interrelationship among different postharvest
factors with regard to the development of flesh-browning. This should also be con-
ducted on other apple varieties and storage conditions.

13.3.3.3  Internal Browning in Pears

Metabolomics- and proteomics-based approaches have been followed to explicate the
physiological and biochemical aspects of browning in “Conference” pears (Pedreschi
et al., 2007, 2008, 2009). The metabolic profiling was carried out on the flesh tissue
of late-harvested “Conference” pears stored in CA containing 1% O2 + 10% CO2 at
–1°C for up to six months, conditions suitable for inducing browning (Pedreschi et al.,
2009). The flesh tissue-browning in pears was mainly related to a disturbed energy
metabolism, alteration in concentrations of metabolites dependent on energy metabo-
lism pathways, a collapsed antioxidant system, and cell-wall architecture. The brown
tissue exhibited a decrease of malic acid and an increase in fumaric acid and gamma
aminobutyric acid (GABA), which indicated a reduced metabolic activity at the level
of the Krebs cycle and a putative block of the GABA shunt pathway. Increased glu-
conic acid concentration might be related to AA degradation due to insufficient reduc-
ing equivalents or to an impaired pentose phosphate pathway. The concentrations of
other compounds, such as trehalose and putrescine, were also considerably higher
in brown (damaged) tissue than in sound tissue, suggesting hypoxic stress. The con-
centration of some sugars, which are typically found in xyloglucans, also increased
during the development of brown tissue, possibly indicating cell-wall breakdown due
to enzymatic processes or chemical reactions of hydroxyl radicals (Pedreschi et al.,
2009). A proteomics study has also shown that down-regulation of ascorbate per-
oxidase (APX), glutathione transferase (GT), and monodehydroascorbate reductase
(MDHAR) in brown tissue was indicative of total impairment of the ascorbate–gluta-
thione cycle (Pedreschi et al., 2007). These studies indicate that development of core
breakdown in “Conference” pears is a consequence of an imbalance between oxida-
tive and reductive processes caused by too-low oxygen or too-high carbon dioxide
conditions, which lead to a deficiency of reducing equivalents for defensive mecha-
nisms, cell-damage repair processes and biosynthesis reactions.

13.3.3.4  Puffing Disorder in Citrus

Puffing disorder in citrus has recently been studied using a combination of transcrip-
tomics and metabolomics tools (Ibáñez et al., 2014). Flavedo, albedo and juice sac
tissues of normal citrus fruits and fruits showing puffing disorder were studied using
metabolomics at three developmental stages. Glycolysis, which is the backbone of
294 Advances in Postharvest Fruit and Vegetable Technology

primary metabolism, appeared to be severely affected by the disorder based on both

transcriptomic and metabolomic results. The concentration of citric acid was signifi-
cantly lower in puffed fruits (Ibáñez et al., 2014). Transcript analysis further showed
that glycolysis and also carbohydrate metabolism were significantly altered in puffed
samples in both albedo and flavedo. Gene expression of invertases and sucrose
exporters, amylose-starch and starch-maltose converters was higher in puffed fruits,
while genes related to gibberellin and cytokinin signaling were down-regulated in
symptomatic albedo tissues, suggesting that these hormones play key roles in the
disorder. The outcomes of this study may be applied toward the development of early
diagnostic methods based on host response genes and metabolites (i.e., citric acid),
and toward therapeutics based on hormones (Ibáñez et al., 2014).

13.3.4 Preharvest Treatments
The targeted profiling of various metabolites in response to preharvest treatments
have the potential to provide greater insights into the understanding of the basic
mechanism of action of these treatments.
Preharvest application of ethephon (2-chloroethylphosphonic acid) on sweet
cheery (Prunus avium L.) has been reported to induce significant shifts in the
metabolome of fruit (Smith et  al., 2011). GC–MS-based metabolic profiling was
conducted on cherry cultivars “Bing,” “Chelan,” and “Skeena” fruit mesocarp and
exocarp tissue to better understand underlying quality-related metabolism associ-
ated with ethephon application. Nearly 200 identified and partially characterized
metabolites from mesocarp and exocarp tissue were characterized and evaluated.
PCA models revealed changes in the metabolome associated with both natural rip-
ening and ethephon-induced changes, including associations to key color, acid, and
sugar components, such as cyanidin 3-glucoside, malic acid, and sugar metabolism.
This metabolomics approach successfully deciphered the effects of ethephon treat-
ment on primary and secondary metabolites that are responsible for color and flavor
attributes of three important cherry cultivars. The overview of the whole metabo-
lome response has the potential to screen the response of different cultivars to such
preharvest treatments aimed at improving fruit quality and harvest practices.
NMR-based global metabolomics approach was followed by Zhang et al. (2012)
to compare the metabolite profiles obtained for “Satsuma” mandarin orange juices
prepared from fruit harvested in eleven separate orchards, to determine whether pre-
harvest orchard factors such as the application of foliar fertilization or pesticides
would significantly alter the metabolite concentration of the juices. The results
showed that both foliar fertilization and pesticides lowered the Brix/acid ratio, and
caused major changes to amino acid levels, as well as levels of other organic mol-
ecules. The change of 1 unit Brix/acid ratio was detected by 68% of subjects in an
untrained sensory panel (Zhang et al., 2012). This study demonstrated that metabo-
lomic analysis may be useful to optimize orchard practices such as fertilizer- and
pesticide-use to obtain an optimal sensory profile.
In a recent study on improvement of litchi pericarp color through preharvest appli-
cation of plant growth regulators (PGR), abscisic acid (ABA) and ethephon, Singh
et  al. (2014) reported that exogenous application of ABA at the color-break stage
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 295

significantly increased the concentration of total anthocyanins and cyanidin-3-O-

rutinoside (the major anthocyanin contributing 71%–96% of the total anthocyanins)
in litchi pericarp, compared to ethephon. Among the different anthocyanins quanti-
fied, the relative contribution of cyanidin-3,5-diglucoside to the total anthocyanins
was significantly higher in all PGR-treated fruit compared to control, but the con-
centration of cyanidin-3-O-glucoside was specifically enhanced by ABA. No signifi-
cant effect on the concentrations of epicatechin, and quercetin-3-O-rutinoside was
observed in response to PGR treatments (Singh et  al., 2014). Ethephon treatment
did not significantly increase the anthocyanins levels in the pericarp, but resulted
in greater degradation of chlorophyll pigments than in the untreated control fruit.
This work is an example of comprehensive profiling of different types of antho-
cyanins, chlorophylls and other flavonoids in the pericarp peel responsible for the
appearance-quality of fruit.

13.3.5 Postharvest Treatments
There is increasing interest in the application of metabolomics alone or in conjunc-
tion with transcriptomics and proteomics to understand the effects of postharvest
treatments on the biological processes in the fruits. The effects of postharvest treat-
ments with 1-MCP and DPA on apple peel metabolome are discussed in the previous
Section 13.3.3.1.
Postharvest heat treatments have been widely studied and used to control a range
of postharvest disorders and are commercially used as quarantine treatments. Perotti
et al. (2011) reported the changes in proteome and metabolome of “Valencia” orange
(Citrus sinensis “cv. Valencia” late) subject to a postharvest heat treatment. The heat
treatment resulted in numerous changes in the general metabolism of orange fruit
in both exocarp and in endocarp, such as alterations in the activities of antioxidant
enzymes, induction of key proteins in response to pathogen attack, changes in com-
pounds involved in major metabolic pathways and possibly a cellular-reorganization
process. All these changes lead to a lower degree of susceptibility of the fruit against
fungal pathogens, while explaining the maintenance of postharvest quality (Perotti
et al., 2011).
Yun et al. (2013) also conducted a proteomic and metabolomic study of mandarin
peel during storage after a two-min. hot-water treatment (HWT) at 52°C. This treat-
ment successfully suppressed the development of Penicillium italicum and reduced
chilling-related injury during storage. The metabolite analysis was performed fol-
lowing a hybrid approach using both GC–MS and LC–QTOF–MS platforms. About
62 metabolites were identified, which were grouped into alcohols, amino acids,
­sugars, organic acids, and fatty acids. Most sugars and fatty acids increased in the
peel after the HWT, but the sugar levels returned to control levels during storage
(Yun et al., 2013). The levels of organic acids were lower immediately after the HWT
but then increased to a higher level than control fruit during storage. Amino acids
were also shown to decrease with the HWT and remained lower than control dur-
ing storage. An exception was ornithine, which was 2.5 times higher than control
fruit. Interestingly, H2O2 content decreased, while lignin content increased in heat-
treated peel compared to the control. This may import increased fruit resistibility in
296 Advances in Postharvest Fruit and Vegetable Technology

response to external stress. In addition flavonoids, substances that are well-known

to be effective in reducing external stress, were shown to be up-regulated in heat-
treated pericarp (Yun et al., 2013).
To prevent or ameliorate chilling-related injuries in peaches, heat treatment is often
applied prior to cold storage. Lauxmann et al. (2014) conducted metabolic profiling to
determine the metabolites’ dynamics associated with the induction of acquired toler-
ance to chilling-related injuries in response to heat-shock. “Dixiland” peach fruits
exposed to a heat treatment, cold stored, or after a combined treatment of heat and
cold, were compared with untreated control fruit ripened at 20°C. Dramatic changes
in the levels of compatible solutes such as galactinol and raffinose were observed,
while amino acid precursors of the phenylpropanoid pathway were also modified due
to the stress treatments, as was the polyamine putrescine (Lauxmann et al., 2014).
The changes in the metabolome of peach during and after heat treatment showed
similarity to those changes observed in citrus by Yun et al. (2013). The sugars and
alcohol sugars were increased in heat-treated fruit and most remained high when fruit
was transferred to 20°C, though fructose and glucose returned to control fruit levels.
Galactinol was the sugar most elevated by heat treatment and remained high if the
fruit was placed in 0°C, but not when the fruit was maintained at 20°C. Organic acids
tended to decrease in heat-treated fruit compared to their levels at harvest. The amino
acid levels in heat-treated peach fruit generally increased either during or after the
treatment. The identification of such key metabolites, which predispose the fruit to
cope with different stress situations, will likely greatly accelerate the design and the
improvement of plant-breeding programs and postharvest treatment protocols.
Quarantine regulations in several mango-importing countries, such as Australia,
Japan, and New Zealand, require the fresh fruit to undergo a postharvest vapor
heat treatment (VHT) in order to be accepted for import. The objective of VHT
is to eliminate the risk of the entry of insect pests associated with the fruit into
the importing country. VHT involves the use of hot air saturated with water vapor
to heat the fruit core to a specified temperature and hold that temperature for a
defined period to ensure that all target insect pests are killed. However, VHT is
known to cause some deleterious effects on fruit quality. Singh and Saini (2014)
reported the influence of postharvest VHT on targeted profiling of aroma volatiles
during fruit-ripening in mango (cv. “Chausa”) using GC–MS. Reversible inhibition
of the emission of aroma volatiles was observed in heat-treated fruit, with a signifi-
cant alteration in the aroma-volatiles profiles at different stages of fruit-ripening.
The heat-induced increase in the rate of fruit-ripening proceeded with a significant
lag in the emission of aroma volatiles. The suppression of aroma volatiles at the
ripe stage in heat-treated fruit might adversely impact the consumers’ acceptance
of fruit. The temporal and quantitative variation in the aroma-volatiles profiles of
mango fruit in response to VHT were observed through this study, and demonstrate
the versatility of these techniques.

13.3.6 Postharvest Storage Conditions

The targeted analysis of a few metabolites responsible for specific quality attributes
such as ethanol and vitamin C content using GC and HPLC techniques, is common
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 297

in many postharvest studies and will not be discussed in this section. The emphasis
of this discussion will focus on the high-throughput targeted profiling of important
metabolites and explore their regulatory roles, using informatics tools that can pro-
vide comprehensive information about the fate of these molecules in response to var-
ious postharvest storage conditions. Matsumoto and Ikoma (2012) recently reported
the application of the LC–MS/MS approach to quantitate sugars, organic acids and
amino acids in the juice sacs of “Satsuma” mandarins (Citrus unshiu Marc. cv.
“Aoshima-unshiu”) stored at 5°C, 10°C, 20°C, and 30°C for 14 days. Without any
significant change in sugars during storage at different temperatures, organic acids
decreased slightly at all temperatures, with the exception of malic acid at 30°C,
which increased slightly. Two amino acids, ornithine and glutamine, increased at
5°C, but they did not increase at other temperatures. The content of 11 amino acids
(phenylalanine, tryptophan, tyrosine, isoleucine, leucine, valine, threonine, lysine,
methionine, histidine, and γ-amino butyric acid) was higher at 20°C and 30°C than
at other temperatures. Moreover, amino acids responded to temperature differently:
two amino acids were cold-responsive, and 11 were heat-responsive. The best tem-
perature to minimize the postharvest changes in amino acid profiles in the juice sacs
was 10°C. This study demonstrates the value of high throughput LC–MS/MS in
clearly defining the biochemical changes associated with storage.

13.3.7 Pre- and Postharvest Diseases

The concept of biomarker discovery is also evolving towards understanding host–
pathogen interactions for disease diagnosis in fruit and vegetables. The selective
volatiles compounds from the headspace have been found useful in the identification
of fungal diseases in apple fruit (Vikram et al., 2004). This approach can lead to the
development of biosensors to monitor the development of disease during postharvest
storage. Preharvest diseases are known to influence fruit quality and postharvest
behavior. Baldwin et al. (2010) performed the targeted analysis of secondary metab-
olites to study the effect of Huanglongbing (HLB) on citrus fruit flavor. The results
showed that asymptomatic fruit from symptomatic trees were similar to healthy fruit
for many of the quality factors measured, but that juice from asymptomatic and,
especially, symptomatic fruits, were often higher in the bitter compounds limonin
and nomilin (Baldwin et  al., 2010). The application of the metabolomics tool has
even been explored to differentiate HLB infection from zinc deficiency in citrus trees
(Cevallos-Cevallos et al., 2011b). HLB is a devastating disease in many parts of the
world, but the symptoms are similar to zinc deficiency, and developing a reliable and
robust method to differentiate these symptoms is a priority for i­ndustry. Cevallos-
Cevallos et  al. (2011b) identified six possible biomarkers for HLB, of which four
were identified as proline, β-elemene, (-) trans-caryophyllene, and α-humulene were
very useful for rapid differentiation of HLB from zinc deficiency. A more recent
study has compared the effects of HLB on the citrus metabolome analysing fruit,
juice from healthy, HLB-asymptomatic, and HLB-symptomatic Hamlin, as well as
from healthy and HLB-symptomatic “Valencia” sweet oranges (Chin et al., 2014).
The NMR-based metabolomics approach revealed differences in the concentration
of several metabolites, including phenylalanine, histidine, limonin, and synephrine,
298 Advances in Postharvest Fruit and Vegetable Technology

between control or asymptomatic fruit and symptomatic fruit, regardless of the

­citrus variety or location. The data indicated that HLB infection presents a strong
metabolic response that is observed across different cultivars and regions, suggesting
the potential for generation of metabolite-based biomarkers of HLB infection.

13.4 METABOLOMICS APPLICATIONS IN
SAFETY OF FRESH PRODUCE
Food safety is an emerging challenge for many horticultural industries. Recent food-
borne outbreaks have raised serious public-health concerns and prompted debate
over the responsibilities and accountabilities of primary producers, food industries,
and regulatory authorities. From the public-health perspective, chemical and micro-
bial hazards are the most common in fresh produce. The chemical hazards related
to the presence of agrochemical residues, such as fungicides, herbicides, and insec-
ticides, on fresh produce, are strictly monitored and regulated by the authorities.
Residue levels in fresh produce tend to decrease after application during any with-
holding period and during postharvest washing and in storage/transit time in the sup-
ply chain. However, the microbial hazards, which include contamination with human
pathogens during preharvest and postharvest stages, tend to exponentially increase
under favorable conditions in the supply chain and pose severe risk to the consumers.
Fresh horticultural produce have recently been implicated in several food-borne ill-
nesses. Some horticultural crops, such as melons, leafy vegetables, berries, nuts, and
tomatoes, are considered high-risk due to their susceptibility to carry and support
proliferation of human pathogens. There are several risk factors during on-farm pro-
duction and postharvest handling of the produce, which predispose horticultural pro-
duce to microbial and chemical hazards. To prevent exposure of consumers to these
risks, risk assessment is a mandatory practice to meet the regulatory requirements
of food-safety authorities. The application of advanced analytical techniques with
high resolution, sensitivity and specificity, constitutes the core of the food-safety
risk-assessment plans for fresh produce. This section will review the recent devel-
opments in application of metabolomics tools to determine the maximum residue
limits (MRL) in fresh produce, to develop tools for ensuring microbial safety and to
address the traceability concerns.

13.4.1 Detection and Quantification of Chemical Residues

The agrichemical residues are among the most important food-safety concerns for
fresh produce. The MRLs on fresh produce have been defined for most pesticides
and toxins by different countries. However, the MRLs are usually at very low lev-
els, requiring highly accurate and sensitive analytical platforms for measurements.
The increasing pressure on food-safety authorities due to public health and envi-
ronmental issues is further pushing the MRLs to extremely low levels. MS-based
approaches have significantly enhanced the detection and quantification limits for
various chemical residues and toxins. These methods are reliant upon the hyphen-
ated techniques involving separation (chromatography) and detection systems (MS).
The separation techniques have evolved in the recent past towards high-throughput
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 299

and analyte-specific column chemistry. For example, ultra-high-performance liquid

chromatography (UHPLC) is emerging as the technique of choice for coupling with
mass spectrometers. The separation of analytes has also been enhanced through two-
dimensional chromatography methods such as 2D–GC and 2D–HPLC. Furthermore,
sample preparation techniques have also been improved to achieve better and selec-
tive recoveries of compounds of interest. A typical sample-preparation protocol for
chemical residues involves three steps: extraction, clean-up and preconcentration.
Solvent extraction and solid-phase extraction (SPE) are the procedures most com-
monly applied. However, the development of the QuEChERS (quick, easy, cheap,
effective, rugged, and safe) protocol is increasingly employed for sample-prepara-
tion before LC–MS applications. This method basically involves an extraction step
with acetonitrile, followed by pH adjustment for phase separation, centrifugation,
and SPE clean-up. Other extraction techniques, such as solid-phase microextraction
(SPME) and stir bar sorptive extraction (SBSE), have been shown to successfully
extract and enrich the compounds of interests from different food matrices. In addi-
tion, the automation of SPME and SBSE techniques (e.g., Gerstel’s Multipurpose
Sampler) has accelerated the adoption and outcomes of these techniques.
The goal of metabolomics applications in chemical residues is to achieve the
highest sensitivity, accuracy and repeatability of results in the shortest possible
analysis time for multiple classes of pesticides in different food matrices. The high-
throughput methods based on GC–MS/MS and LC–MS/MS have been developed
and commercially adopted for determination of pesticide residues in fresh produce.
Camino-Sánchez et al. (2011) reported a rapid, sensitive, accurate and reliable mul-
tiresidue method for quantification and confirmation of 121 common agricultural
pesticides in fruits and vegetables by GC–EI–MS/MS (QqQ in SRM mode). This
method was accredited according to International Standard UNE-EN ISO/IEC
17025:2005 and was validated and applied to 1463 vegetable and fruit samples col-
lected over one year from extensive greenhouse cultures in Spain. This method
separated the pesticides in less than 30 min. and the limit of quantitation was
<10 µg kg−1. The results showed that only three pepper samples, one of tomato and
one of cucumber had residues above the MRL (0.01 mg kg−1) according to European
Union directives (Camino-Sánchez et al., 2011). The development of the multiresidue
analytical method based on GC–EI–MS/MS was further extended by Banerjee et al.
(2012) when they reported an excellent method for the simultaneous estimation of
375 organic contaminants, including 349 pesticides, 11 polychlorinated biphenyls
(PCBs), and 15 polyaromatic hydrocarbons (PAHs), extracted from grape, pome-
granate, okra, tomato, and onion matrices. The GC–EI–MS/MS parameters were
optimized for analysis of all the 375 compounds within a 40 min run-time with limit
of quantification for most of the compounds at <10 μg L −1, which is well below their
respective European Union MRLs.
Núñez et al. (2012) developed a method based on LC–MS and LC–MS/MS for
the confirmation of a group of 100 pesticides (herbicides, insecticides, and fungi-
cides) in fruits and vegetables. LC–MS/MS—using highly-selective selected reac-
tion monitoring (H-SRM) acquisition mode, monitoring two transitions for each
compound—was reported to be the most sensitive methodology. Limits of detection
(by acquiring two transitions and with the ion-ratio requirements) ranged between
300 Advances in Postharvest Fruit and Vegetable Technology

0.01 and 20 μg kg−1 were obtained. This method could meet sensitivity requirements
for the MRLs established by the European Union regulation for food monitoring
programs. The LC–MS and LC–MS/MS strategies developed were successfully
applied for the analysis and confirmation of pesticides in different types of fruit
and vegetables samples. In addition to these general pesticides-residue-testing meth-
ods, there are some reports on commodity-specific analytical methods for various
pesticides. For example, Fleury-Filho et al. (2012) developed a highly selective and
sensitive method based on LC–MS/MS for the simultaneous determination of 98
pesticide residues in mango fruit. Similarly, Banerjee et al. (2013) developed a GC–
MS method for multiresidue determination of 47 pesticides in grapes with limit of
quantifications of each compound in compliance with the EU MRL requirements.
This method was successfully applied for analysis of real-world samples for incurred
residues.
Methods based on QqQ–MS have been most commonly adopted for multiresidue
analysis in fresh produce, as these allow identification of target residue and quanti-
fication with an adequate concentration range and reproducibility in complex food
matrices at extremely low levels (ng kg−1). On the other hand, the high resolution
mass spectrometers (HRMS) systems provide high resolution, accurate mass and
high full-scan sensitivity and selectivity, making them attractive for residue analysis
in food (Gómez-Ramos et al., 2013). They allow operation for both target and non-
target compounds, and options to work in full-scan mode or MS/MS mode, and in
sequential or simultaneous mode. This translates into a rapid, effective option for
routine laboratories. The drawbacks with the HRMS system, such as accurate mass
data obtained above 5 ppm, lower resolving powers than 20,000, saturation effects
with certain compounds, and short dynamic range, have been now addressed in the
advanced systems to greater extent (Gómez-Ramos et al., 2013). Some studies have
shown the quantitative capabilities of HRMS instruments to QqQ systems, and are
valuable indicators of the potential of LC–TOF–MS for large-scale quantitative mul-
tiresidue analysis. Taylor et al. (2008) evaluated the potential of UPLC–TOF–MS
for the quantitative analysis of 100 pesticides in strawberry fruit and the results were
compared with those obtained using an UPLC–MS/MS MRM. Residues found in
the samples ranged from 0.025 to 0.28 mg kg−1 and they were in excellent agreement
with results obtained using UPLC–MS/MS. Lacina et  al. (2010) investigated the
potential of UPLC–TOF–MS in the analysis of 212 pesticides in apple, strawberry,
spinach, and tomato. Compared in tandem with mass analyzers such as QqQ, the
sensitivity of TOF is lower and the linear dynamic range of the studied instrument
is rather narrower. The detailed account of application of metabolomics tools, based
on LC–MS/MS, for pesticide residues in fresh produce, can be obtained from the
reviews published by Castro-Puyana and Herrero (2013) and Gómez-Ramos et al.
(2013).

13.4.2 Detection of Mycotoxins
Mycotoxins are secondary metabolites produced by various fungi on a broad range of
food, including fresh or dried horticultural fruit and vegetables. These mycotoxins are
capable of causing acute toxic, carcinogenic, mutagenic, teratogenic, immunotoxic,
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 301

or oestrogenic effects in animals and humans. Considering their potential to cause

toxicity, maximum concentrations of mycotoxins permissible in food are defined by
regulatory authorities to prevent consumer exposure to them. The most important
mycotoxins in food and feed, which are regulated, are aflatoxins (AFB1, AFB2,
AFG1, AFG2), ochratoxin A (OTA), type A and B trichothecenes (e.g., HT-2 toxin,
T-2 toxin and deoxynivalenol (DON)), fumonisins, and zearalenone. The multitarget
analytical methods have replaced the single analyte methods due to their versatil-
ity and throughput (Ren et al., 2007; Sulyok et al., 2007; Spanjer et al., 2008). New
metabolomics-based methods have been recently developed and validated in order to
identify, quantify/semi-quantify the levels of mycotoxins in different foods and nuts.
Varga et al. (2013) reported a multitarget method for the determination of 191 fungal
metabolites in almonds, hazelnuts, peanuts, and pistachios. The method included all
mycotoxins regularly found in food, and the mycotoxins regulated in the European
Union. The applicability of the developed method was demonstrated through the
analysis of 53 naturally-contaminated nut samples from Austria and Turkey. Varga
et al. (2013) showed that of the 40 toxins that were quantified; the most frequently
found mycotoxins were beauvericin (79%), enniatin B (62%) and macrosporin (57%).
In the most contaminated hazelnut sample, 26 different fungal metabolites were
detected. The application of UPLC-MS/MS has also been extended to quantitate 26
mycotoxins in finished grain and nut products (Liao et al., 2013).
Patulin is another very important mycotoxin in apple and apple-derived products
such as apple juice, cider and puree. It has also been reported from other fresh fruits
and fruit-derived products such as blueberry, cranberry, raspberry syrup, and grape
juice. A recent study has shown that patulin can be found on moldy fresh fruits such
as tomatoes, bell pepper, and soft red fruits (Van de Perre et al., 2014). Patulin is
produced by more than 60 fungal species, of which Penicillium expansum (blue
mould) is the most important and often involved in postharvest rots of fresh fruits.
The Codex Alimentarius recommends levels of patulin in fruits and fruit juices to
be lower than 0.05 mg kg−1. In 2006, the European Commission established the fol-
lowing maximum levels of patulin in apple products: 0.05 mg kg−1 for fruit juices
and other drinks derived from apple or apple juice; 0.025 mg kg−1 for solid apple
products; and, 0.01 mg kg−1 for apple products intended for infants and young chil-
dren, and baby foods that are not cereals-based products. Therefore, the estimation
of patulin at such low concentrations is a challenging task for commercial testing
laboratories. Most multitarget analytical methods developed for mycotoxins have
not included patulin among the targets. This is primarily due to its high polarity and
low molecular mass, which commonly lead to low recoveries and/or low sensitivity,
hampering its determination at the regulatory levels.
There are several methods available for the determination of patulin in different
food products, including a multitarget method for mycotoxins developed by Varga
et al. (2013). Recently, a sensitive, quick and reliable method based on UPLC–MS/
MS has been reported to estimate patulin content in fruit samples (Beltrán et  al.,
2014). In this method, chromatographic separation was achieved in less than 4 min.
The comparison of the ion sources showed that the use of ESI caused strong signal-
suppression in samples; however, matrix effect was negligible using APCI, allow-
ing quantification with calibration standards prepared in solvent. The method was
302 Advances in Postharvest Fruit and Vegetable Technology

validated in four different apple matrices (juice, fruit, puree, and compote) at two
concentrations at the low μg kg−1 level (Beltrán et al., 2014). In addition, a GC–MS
based method with a QuEChERS procedure for determination of patulin in apple
juices, has been developed and validated by Kharandi et al. (2013). However, this
method still requires a derivatization procedure using N,O-bis-trimethylsilyl tri-
fluoroacetamide, which is time-consuming. There is further scope to develop new
techniques to provide analytical solutions for mycotoxins widely differing in their
polarity.

13.4.3 Detection of Pathogens
Rapid detection of food-borne pathogens is critical to reduce food-related dis-
ease outbreaks and product recalls. Most important pathogenic bacteria, such as
Salmonella, Escherichia coli OH157: H7 and Listeria monocytogenes, have the
potential to contaminate fresh horticultural produce during production and post-
harvest handling. Therefore, the application of diagnostic tools to detect and iden-
tify these pathogens within a short span of time is a challenging task for the food
­industry. Detection of pathogens using traditional plating technologies takes many
days, while rapid technologies based on antibody or polymerase chain reaction (PCR)
detection usually yield results in less than 48 h. The genomic technologies such as
PCR, real-time PCR, and even next-generation sequencing technologies, have the
potential to replace the conventional phenotyping approaches (Lauri and Mariani,
2009). In addition, proteomics tools using MALDI–TOF have also been employed
to identify and characterize the pathogenic bacterial strains (Barbuddhe et al., 2008;
Dieckmann and Malorny, 2011). The discussion of these technologies in food safety
is beyond the scope of this chapter.
With regard to metabolomics, some attempts have been made to apply MS
tools to identify the pathogenic bacteria in food, but there are no current reports
on such applications in fresh horticultural produce. A metabolomic-based method
for rapid detection of Escherichia coli O157:H7, Salmonella hartford, Salmonella
typhimurium, and Salmonella  ​muenchen in nonselective media was developed
(Cevallos-Cevallos et  al., 2011a). PCA discriminated pathogenic microorganisms
grown in culture media and the metabolites responsible of PCA classification were
dextrose, cadaverine, the aminoacids l-histidine, glycine, and l-tyrosine, as well as
the volatiles 1-octanol, 1-propanol, 1 butanol, 2-ethyl-1-hexanol, and 2,5-dimethyl-
pyrazine. Partial least square (PLS) models, based on the overall metabolite profile
of each bacterium, were able to detect the presence of Escherichia coli O157:H7
and Salmonella spp. at levels of approximately 7 ± 2 CFU/25 g of ground beef and
chicken within 18 h. This research proposed an alternative approach to rapidly
detect two major pathogens based on detecting changes in the metabolite profile
during incubation in nonselective culture media (Cevallos-Cevallos et al., 2011a).
Another recent study has described a proof-of-concept application of a metabolo-
mic technique for the rapid detection of Listeria, applied to nutrient media and a
complex food sample (milk) inoculated with a pathogenic Listeria strain (L. mono-
cytogenes). It was found that a profile of intracellular and extracellular metabo-
lites associated with L. monocytogenes could be obtained using GC coupled to
Metabolomics Tools for Postharvest Quality and Safety of Fresh Produce 303

orthogonal acceleration TOF–MS. Chemometric analysis showed that it is possible

to differentiate between the uninoculated samples and samples inoculated with
Listeria based on L. monocytogenes metabolic activity. This research demonstrates
that metabolomics has the potential for rapidly identifying food contaminated with
Listeria and could provide a means for enhancing monitoring programs and ensur-
ing food safety (Beale et al., 2014). The outcomes of these studies are not directly
transferable to an industrial environment, but these proofs of concept provide evi-
dence of potential application and extension to other food commodities, such as
fresh fruit and vegetables.

13.4.4 Traceability, Fraud, and Malpractices

Traceability of fresh produce has now become an essential requirement for the trade
of fresh produce. It requires regulation and monitoring of the entire supply chain,
allowing the food to be traced through every step of its production back to its origin.
Labeling is an integral component of traceability that enables the identification of the
produce through complex supply-chain systems. The proper label indicating coun-
try/region of origin, and production system (organic or integrated) of the produce
not only informs the consumer, but also helps the food industry in fetching premium
value of produce linked to some geographical indicators. Deliberate or accidental
mislabeling, which can mislead the consumers, constitutes food fraud, as per the
food safety and standards regulations of many countries. Furthermore, in the event
of food-borne outbreaks and recalls, proper labeling facilitates the consumers as
well as enforcement agencies to trace the background of the food during investiga-
tions and legal procedures.
Metabolomics tools have the potential to discover biomarkers for fresh produce
linked to geographical origin and production systems. The discovery of markers
having discriminating powers to authenticate the information on labels seems to
be the future of research at this stage. The research in this direction has been quite
slow and limited to some commodities such as olive oil, tea, coffee, and herbal prod-
ucts, but this area of application is also gaining momentum. The elemental compo-
sitions and stable isotope ratios of different fruits and nuts (Anderson and Smith,
2005; Perez et al., 2006) determined by inductively-coupled plasma MS showed the
potential for “ionomics” to play an important role in traceability. A comprehensive
review on traceability markers of olive oils has been presented by Montealegre et al.
(2010) and highlighted the then state-of-art research using different approaches of
genomics, chemometry, and ionomics. The high-resolution-magic-angle spinning–
NMR (HRMAS–NMR) spectroscopy approach has been recently found to discrimi-
nate potato tubers grown under organic versus conventional management systems
(Pacifico et al., 2013). Nitrogen content in organic potatoes decreased by 11%–14%
and GABA and lysine accumulated in the organic tubers. For the first time, this
study provided the basis for constructing a validated and robust experimental model
that can efficiently distinguish potato tubers according to the cultivation system.
However the metabolite-based markers linked to different types of production sys-
tems and production region have not been robustly tested and validated at large
scales to bring them into commercial practice.
304 Advances in Postharvest Fruit and Vegetable Technology

Metabolomic tools can also be applied to confirm the use of banned/prohibited

substances during production and postharvest handling of fresh produce. Though
most agrichemical residues can now be easily detected and quantified using metabo-
lomics tools, there are still some gaps that need attention. For example, the use of
calcium carbide for postharvest fruit-ripening is prohibited by law in several coun-
tries, including India. However, this compound has been continuously used for rip-
ening mango, banana and papaya fruit, as the regulatory authorities are unable to
distinguish the fruit ripened with ethylene and calcium carbide. The method of appli-
cation of carbide barely leaves any residue on the fruit’s surface as the powder, when
comes in contact with moisture, liberates acetylene gas, which mimics the action of
ethylene to induce fruit-ripening. Metabolomics approaches, using both GC–MS and
LC–QTOF–MS techniques, have shown promising results to distinguish the fruit
ripened with carbide and ethylene (Singh unpublished).

13.5 CONCLUSIONS
As metabolites are considered the downstream products of cellular regulatory
processes, metabolomics data can precisely characterize cells, tissues, or whole
organisms, by defining specific biochemical phenotypes that are representative
of physiological or developmental states. The metabolomics approach is consid-
ered superior to other “omics” tools, which are often compromised with biological
enigmas such as epigenetic modifications, posttranscriptional and posttranslational
changes. The metabolites are the ultimate signatures of a genome, offering strong
correlations with phenotypes and chemotypes. In plant and food systems, metabolo-
mics are beginning to be applied to gain better understanding of various pathways
and systems involved in improvement of horticultural produce for better nutrition,
processing and postharvest storage and quality. The metabolomics applications have
been extended to define and measure comprehensive quality traits in various food
crops, to ensure food safety aspects (adulterants, contaminants, allergens, etc.), and
to develop quality and processing standards for regulatory authorities. In the mod-
ern era, the definition of quality is expanding beyond a few parameters to multiple
parameters determined using sophisticated analytical platforms to achieve excel-
lence in developing new product(s) with highest standards of quality and safety.

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 14 Recent Developments
in Proteomic
Analysis of Fruits
Jun Song

CONTENTS
14.1 Introduction...................................................................................................309
14.2 Sample Preparation........................................................................................ 310
14.3 Analytical Proteomic Platforms.................................................................... 312
14.3.1 2-DE-Based Comparative Proteomics............................................... 312
14.3.2 Nongel-Based Proteomics.................................................................. 314
14.3.2.1 Label-Free Approaches....................................................... 314
14.3.2.2 Labeling and Targeted Quantitative Proteomic
Approaches......................................................................... 314
14.3.3 Informatics/Bioinformatics............................................................... 320
14.4 Proteomic Research on Apple and Pear........................................................ 321
14.5 Future Research Perspectives........................................................................ 323
Acknowledgments................................................................................................... 324
References............................................................................................................... 324

14.1 INTRODUCTION
Proteomics is the study of “the entire protein complement expressed by a genome in
a cell or tissue type” (Pandey and Mann, 2004). Proteomics now covers all aspects
of protein research in a cell, encompassing the abundance and changes in time and
biological behavior, as well as posttranslational modifications (PTMs) related to the
biological functions and protein–protein interactions. Proteomics has become the
crucial element of “omic” approaches or system biology tools, and is contributing to
a new understanding of biological systems.
Fruit and vegetable quality is the result of combination of multiple attributes
including appearance, flavor (taste and aroma), and nutritional components (Kader,
2004). Significant research efforts on both pre- and postharvest technologies aim to
improve and maintain eating quality and nutritional content. Although there have
been many successful postharvest applications implemented on many fruit and veg-
etables, there is little fundamental understanding of the gene, protein, and metabo-
lite networks regulating the quality of fruits and vegetables. Genomic research has
recently been applied on fruit and vegetables, and has begun to identify genes related

309
310 Advances in Postharvest Fruit and Vegetable Technology

to ripening and quality (Giovannoni, 2004). However, many of the biosynthetic path-
ways associated with fruit and vegetable quality are not fully understood (Song and
Forney, 2008).
A common technique used in proteomics is the examination of differences
between pairs of samples, such as produce at different developmental stages, held
under different abiotic stress conditions, or between diseased and healthy tissue.
Proteomics can be extended to examine the fundamental changes during postharvest
handling, and storage. Research platforms employed in proteomic studies generally
utilize high-resolution mass spectrometry to directly study the proteins and their
modifications (top-down), or study the peptides digested from proteins, using liquid
chromatograph and mass spectrometry (LC/MS), equipped with nanospray ioniza-
tion source (bottom-up).
The early development of protein analysis has been challenging, where the results
were incomplete, or were merely qualitative. In addition, it is well-known that changes
in protein abundance may not always be coincident with gene expression data, or from
DNA microarray, as mRNA abundance poorly correlates with protein abundance
(Baginsky et al., 2010). However, with recent developments and advances in genome
sequencing, considerable progress has been achieved with MS-based proteomics, and
many proteins have been identified. MS-based proteomics have allowed the identifi-
cation and quantitative profiling of organism proteomes, and the systematic analysis
of protein modifications, and protein–protein interactions. All these developments
have offered a new range of opportunities for geneticists and network biologists to
improve existing models of how phenotypes emerge (Gstaiger and Aebersold, 2009).
Ideally, proteomic studies should identify and quantify all the proteins in a cell. With
the published data-base of Arabidopsis thaliana, more than 227,396 proteins have
been listed from ca. 30,000 protein coding genes (NCBI: http://www.ncbi.nlm.nih.
gov/, accessed on April 2014). However, the number of proteins that can be inves-
tigated in a single proteomic study, remains limited. As a classical proteomic sepa-
ration platform, two-dimensional electrophoresis (2-DE), along with MS, has been
applied to resolve hundreds/thousands of proteins, facilitating peptide composition
analysis, sequencing, and polypeptide identification. 2-DE is especially useful for
comparative studies between pairs of samples, and has been the dominant technique
applied to fruits and vegetables, together with sample preparation, and protocol estab-
lishment. Significant improvements in proteomic research platforms have been made
from a qualitative platform (2-DE) to techniques of a more quantitative nature (nongel
based), especially with model systems such as human tissues and yeast.
In this chapter, only qualitative and quantitative analysis of proteins employing
LC/MS on peptides, will be discussed. Readers can find detailed information on
top-down proteomic research platforms in other reviews (Parks et al., 2007; Zhou
et al., 2012). Data will also be presented on the potential application to fruit such as
apples and pears.

14.2  SAMPLE PREPARATION

In a proteomic study, a major challenge is how to isolate as many proteins as pos-
sible from a biological sample, and in a form that is suitable for downstream analysis.
Recent Developments in Proteomic Analysis of Fruits 311

Fruits are considered recalcitrant plant tissues for proteomic analysis, due to rela-
tively low protein content, and the presence of numerous interfering substances, such
as pigments, carbohydrates, polyphenols, polysaccharides, and starch (Saravanan
and Rose, 2004). Significant research has optimized sample preparation procedures
from sample handling, protein extraction, and solubilization on young immature
vegetative tissues (Gallardo et al., 2002; Jacobs et al., 2005). However, many of these
protocols have been shown to be unsuitable and not applicable to mature fruit sam-
ples (Song et al., 2006).
As a general procedure, plant tissues are ground to a powder in liquid nitrogen,
followed by acetone or acetone containing trichloroacetic acid for precipitation of
proteins, which are then solubilized in a suitable buffer (Mechin et al., 2003; Wang
et al., 2003). The precipitation agents are very important, where both acetone (with
and without TCA) as well as methanol/ammonium acetate (with dithiothreitol,
DTT) are widely used precipitation agents. In a study to improve proteomic proce-
dures, Saravanan and Rose found no significant difference between TCA/acetone
and phenol-based extraction procedures. However, they reported that the phenol
extraction was better in removing the interfering compounds (Saravanan and Rose,
2004).
Another study improved the protein extraction protocol for mature fruit, using a
protocol developed specifically for apple and banana fruit, that used a heated extrac-
tion buffer containing sodium dodecyl sulfate (SDS), DTT, glycerol, and Tris-HCl
buffer (pH 8.5) (Song et al., 2006). This study showed that the SDS was useful in
enhancing protein solubility, but then it had to be removed from the sample prior to
LC/MS analysis, through acetone precipitation (Song et al., 2006).
It is well-known that phenol can be useful in the protein extraction procedure,
and it is especially beneficial for the extraction of low concentrations of protein in
vegetative plant tissues (Hurkman and Tanaka, 1986). Saravanan and Rose (2004)
showed that a phenol extraction procedure—of protein from tomato and banana
fruit—was comparable to protein precipitation with acetone, with an increase in
protein spots and glycoproteins (Saravanan and Rose, 2004). However, Carpentier
et al. (2005) found that TCA/acetone precipitation, and phenol extraction, gave com-
parable results on banana, apple, and potato plant tissues. Zheng et al. (2007) fur-
ther showed that phenol protocol resulted in significantly higher protein yields from
apple and strawberry fruit, and, through 2-DE analysis of apple protein extracts,
revealed 1422 protein spots associated with the phenol protocol, and 849 spots
with the SDS protocol. Zheng et al. (2007) suggested that a phenol-based protein
extraction protocol should be used as a standard procedure for most fruit tissues.
However, Wang et al. (2006) showed that the TCA/acetone and methanol washes,
combined with a phenol/SDS protein extraction procedure, are more efficient than
phenol and SDS alone (Wang et al., 2006). While Pedreschi et al. (2007) reported
that methanol/ammonium acetate precipitation with DTT, and no PVPP at pH 8.0,
would be the best procedure for protein extraction from pear fruit. Subsequent solu-
bilization of the protein extract is very important to the downstream protein analy-
sis. Solubilization requires the proteins to be dissolved and displayed on the gels,
to generate discrete protein spots that are suitable for further MS analysis. Among
the early stages of fruit proteomic studies, contributors to the development and
312 Advances in Postharvest Fruit and Vegetable Technology

optimization of fruit protein sample preparation procedures (Carpentier et al., 2005;

Rose et al., 2004; Song et al., 2006; Wang et al., 2006; Zheng et al., 2007). The
protein extraction methods developed for 2-DE are generally suitable for nongel-
based approaches, using LC/MS, but SDS and CHAPS (3-[(3-Cholamidopropyl)
dimethylammonio]-1-propanesulfonate) must be avoided, because they interfere
with downstream LC/MS analysis. Most nongel-based proteomic technologies have
been developed on human tissues, yeast, and bacteria, but these can be directly
applied on fruit and plant samples.

14.3  ANALYTICAL PROTEOMIC PLATFORMS

14.3.1  2-DE-Based Comparative Proteomics
A gel-based 2-dimensional electrophoresis (2-DE) platform has been developed to
separate proteins by their isoelectric point and size. This system has continually
been improved with better resolution, as a result of the development of stable immo-
bilized pH gradients (IPG)-based systems, which have considerable resolving power
and reproducibility (Görg et al., 2004). In general, 2-DE based proteomic procedure
includes isoelectric focusing (IEF, first dimension), sodium dodecyl sulphate poly-
acrylamide gel electrophoresis (SDS-PAGE) separation (second dimension), protein
visualization and detection, protein identification, protein quantitation, and bioin-
formatics (Wittmann-Liebold et al., 2006). 2-DE-based proteomic techniques have
shown to successfully investigate the presence and changes of thousands of proteins.
With the development and application of mass spectrometry (MS) techniques to
determine the molecular weight of proteins and peptides, and continuing completion
of many genomic sequence databases of plants (include some fruit), to match with the
masses, it has become possible, using MS platform, to conduct comprehensive and
high throughput proteomic experiments on identification and quantitation of plant
proteins (Newton et al., 2004; Roberts, 2002). Although 2-DE has limited ability to
detect low abundance proteins, as well as membrane proteins, and narrow dynamic
range of detection, due to the technical difficulties of gel electrophoresis, 2-DE has
been widely used as one of the significant proteomic platforms on many fruit.
The detection and quantitation of proteins is through visualization, by stain-
ing. Radioactive labeling, nonfluorescent stains such as Coomassie Brilliant Blue™
(CBB), silver staining, or fluorescent stains, can be used as staining agents to visual-
ize the proteins on 2-D gel, and create digital images, which can then be quantita-
tively analyzed, using specially designed software. Silver is a very sensitive staining
agent and is MS-compatible, but its use has been limited due to its narrow dynamic
range, prone to interferences, multistep process, and gel-to-gel variation (White
et al., 2004). When different stains were compared, it was reported that both sil-
ver and fluorescent stains are more sensitive than CBB, with better dynamic range
(Harris et al., 2007). Applications of fluorescent stains have become attractive due
to their sensitivity and wider dynamic range of protein concentrations (Nishihara
and Champion, 2002; Patton, 2000). Many fluorescent stains, such as Sypro Ruby,
Deep Purple, RuTBs (ruthenium II-tris(bathophenan-throline dissulfonate), and
Sulforhodamine G fluorescent stains, have been developed, and are commercially
Recent Developments in Proteomic Analysis of Fruits 313

available, with good sensitivity (Lamanda et  al., 2004; Lanne, 2004; Mackintosh
et al., 2003), and no major differences, in terms of protein detention, in most protein
samples, have been reported (Wheelock et al., 2006). Harris et al. (2007) found that
among the five commercially available fluorescent stains, Sypro Ruby was shown to
be superior to others, with high detection of total number of spots, and the lowest
signal-to-noise ratio (Harris et al., 2007). First dimension separation of proteins on
IEF strips requires that the proteins should be adequately resolved, and separated, as
much as possible. In order to improve the resolution to separate thousands of proteins,
18 or 24 cm-long Immobiline™ IEF drystrips, and large format gels (18 × 24 cm),
have become the standard size to increase the protein loads and efficiency. On the
other hand, narrow pH range gradient strips can also be employed to extend the IEF
efficiency (Görg et al., 2004). Under current commercial and physical limitations,
most 2-DE gel electrophoresis have been conducted on IEF strips up to 24 cm in
length, with a 3–11 linear or nonlinear pH range, and sequential SDS-PAGE at 21 cm
long. A large scale gel system with combined multi-pI range, and multiple line SDS/
PAGE gels, has been shown to improve the resolution which resulted in resolving of
11,000 proteins spots (Inagaki and Katsuta, 2004). Routine use of this platform type
may be difficult, but it shows the possibilities for further technical improvements to
2-DE based technologies (Wu et al., 2008).
To overcome the chemical and physical limitations of protein separation by pI
and molecular mass, using IEF strips and 2-DE, a third-dimensional electrophore-
sis can be applied. The use of the difference gel electrophoresis (DIGE) technique
has improved quantitation, and minimized the gel-to-gel variation, in comparative
proteomic research (Tonge et al., 2001). Before mixing the samples and 2-DE sepa-
ration, the protein samples are labeled with three fluorophores Cy2, Cy3, and Cy5
stains, with the covalent labeling on lysine residues. After 2-DE separation, the pro-
teins are scanned with a fluorescence scanner at different wavelengths specific to
each dye, to reveal the different proteomes. To control the variation, one of the stains
is used as internal standard, which then is used in a mix of all the experimental sam-
ples (treated, and control). While other two dyes will be used to differentially label
the treated and control samples, all three labeled samples are then separated on the
same gels (Tonge et al., 2001). The DIGE technique has been applied on several plant
proteomic studies, such as Arapidopsis (Dunkley et  al., 2006), and poplar leaves
(Bohler et al., 2007). Further improvement in the use of the DIGE technique is with
the use of an Alexa-labeled internal standard (ALIS) spiked into protein samples
(Wheelock et al., 2006). The ALIS normalization method is based on incorporation
of an identical protein standard into every sample that is spectrally separated from
the sample to be analyzed. This makes the method robust, and resistant to the overall
changes in protein abundance (Wheelock et al., 2006).
Mass spectrometry has become the central technological platform for proteomic
research. Depending on the mass spectrometry for protein spot identification, either
peptide mass fingerprint (PMF) from a MALDI-TOF (time of flight MS), or frag-
ment of MS (MS/MS spectra), which are produced in the collision cell within the
mass spectrometer to generate the MS/MS spectra can be applied. Both PMF and
PFF rely on the protein sequences in order to interpret spectra (Palagi et al., 2006).
For the sequences of peptides from MS/MS, without known sequences, de novo
314 Advances in Postharvest Fruit and Vegetable Technology

sequencing approach can then be applied (Palagi et al., 2006). Using an apple aller-
gen protein (mal d1) as an example, the 2-DE gel-based procedure followed by a LC/
MS analysis of digested proteins (peptides) with MS/MS spectra, has been illus-
trated in Figure 14.1.

14.3.2 Nongel-Based Proteomics
Nongel-based proteomic techniques are based on either label-free or different
labeling technologies, which allow large-scale quantitative proteomic research on
protein populations, and are believed to overcome the shortcomings of gel-based
technologies.

14.3.2.1  Label-Free Approaches

To overcome the technical challenges in 1D and 2D electrophoresis, a higher reso-
lution and higher capacity 2D separation has been achieved, with an in-line sys-
tem using a biphasic nanocolumn, known as multidimensional protein identification
(MudPIT) (Link et al., 1999). This technique uses a 3–5 cm section of strong cation
exchange resin, upstream from a C18 resin, in a nanocolumn, to allow the accumula-
tion of a large amount of protein, that is released onto the C18 column, with an elu-
ent with an increasing salt concentration. This technique increases the number of
digested proteins that can be identified, and enhances the detection of low abundance
proteins in the mixture (Delahunty and Yates, 2005; Washburn et al., 2002).
In such a label-free protocol, no label is required to be induced to the samples,
and the protein digest is directly analyzed on a mass spectrometer, coupled with the
nanospray LC separation. In general, the acquisition of LC/MS/MS data is only for
the most abundant peptides peaks at each time point (data dependent acquisition).
Therefore, the label-free technique for protein quantitation consists of two differ-
ent strategies; either spectra counting, or intensity-based measurement of peptides.
Spectra-counting is widely used to count the number of MS/MS spectra, acquired
for a certain protein, and, thereby, to estimate the quantity of the protein. While
peptide-intensity can be measured by their precursor ions, it is used as a quantitative
measure of the quantity of peptides. Despite the inexpensive and easy nature, the
label-free platform requires a high resolution mass spectrometer, and is more sensi-
tive to technical variations of LC/MS runs. An additional technique called MSe on
the qTOF MS enables the detection of peptides, precursors, ions, and fragment ions
(MS/MS), using different collision energy from 10 eV to 35 eV (Silva et al., 2006).
A linear relationship between the MS signals, from three most intensive peptides and
proteins, was found (Silva et al., 2006). An excellent review of label-free proteomic
techniques is presented by Levin and Sabine (2010), Matros et al. (2011).

14.3.2.2  Labeling and Targeted Quantitative Proteomic Approaches

The labeled quantitative proteomic platform has been widely applied, and allows
quantitative comparison between/among biological samples, which are differentially
chemical tagged proteins or peptides. Isotope Coded Affinity Tag labeling (iCAT)
uses heavy versus light isotope coded tags, in two comparable samples. This tech-
nique allows differential chemical tagging of proteins from different samples, by
(a) (b)
kDa TIC: from Sample 1 (Jan 19 AE ED13 Spot 6126) of DataJan 19 AE E D13 spot 6126.wiff (Nanospray) Max. 4.9e9 cps
24.85
4.9e9
75 – 4.8e9
4.6e9 1
50 – 4.4e9
4.2e9
4.0e9
3.8e9 22.24
37 –
3.6e9
3.4e9
3.2e9
25 – 3.0e9
2.8e9
2.6e9
2.4e9
2.2e9

Intensity, cps
20 –
2.0e9
1.8e9
1.6e9
1.4e9
1.2e9 2
15 –
62.60 65.11
1.0e9 49.17
8.0e8 47.06 78.53 81.28
67.05 70.56
76.14
6.0e8 31.04 32.76 42.94 49.73
4.0e8 38.30 61.39 83.72
2.0e8
0.0
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Recent Developments in Proteomic Analysis of Fruits

Time, min

FIGURE 14.1  Illustration of spot identification from a 2D gel of apple fruit employing LC/MS. Spot 6126 identified from 2D gel (a) was excised and
digested with trypsine and corresponding peptides were analyzed on a LC/MS system equipped with a nanospray ion source. Total ion chromatography
is shown in (b). During LC/MS analysis, two peptides were detected and their MS/MS spectra were collected at retention time of 24.45 and 29.60 min,
respectively (c) and (d). The MS/MS ion search employing MASCOT revealed doubly charged GAEILEGNGGPGTIK and IFTGEGSQYGYVK,
respectively as peptide 1 and 2. The spot no. 6126 was therefore identified as major allergen mal d1 protein (MW: 17,69 kDa, pI 5.67). More detailed
information can be obtained. (From Zheng, Q. et al. 2007. J. Agric. Food Chem. 55:1663–1673.)
(Continued)
315
 316

(c) +EPI (742.30) Charge (+2) CE (41.6612) CES (10) FT (100): Exp 3, 26.109 min from Sample 1 (Jan 19 AE ED13 Spot 6126) of... Max. 7.0e5 cps
(d) +EPI (724.79) Charge (+2) CE (40.891) CES (10) FT (100): Exp 3, 29.073 min from Sample 1 (Jan 19 AE ED13 Spot 6126) of ... Max. 8.3e5 cps

800.4 901.3
7.0e5 8.3e5
8.0e5
6.5e5
7.5e5
6.0e5 7.0e5

5.5e5 6.5e5
724.8
929.3 6.0e5
5.0e5
1087.4
5.5e5 617.9
4.5e5
5.0e5
743.4
4.0e5
329.2 733.6 4.5e5
3.5e5 466.2
4.0e5

Intensity, cps
424.2

Intensity, cps
3.0e5 3.5e5 629.2 1234.3
312.0 515.3 1042.4
2.5e5 3.0e5
715.7
629.3 2.5e5 844.4
2.0e5
397.1 667.1 384.1 409.1 639.2
2.0e5
1.5e5 548.3
361.2 537.4 611.4 684.4 911.3 1284.6
1.5e5 299.0 373.2 483.2 609.2 740.0 1030.6
379.1 488.3 510.2 601.3 637.4 724.8 782.2
1.0e5 339.0 401.2 418.1 512.2 757.2 1303.6
350.9 442.4 466.4 555.6 586.4 617.2 893.2 1007.4 1155.5 1267.4
1.0e5
581.2 644.2 785.3 883.3 924.0 1185.7 1284.6 1335.1
5.0e4 5.0e4

0.0 0.0
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
m/z, Da m/z, Da

FIGURE 14.1 (Continued)  Illustration of spot identification from a 2D gel of apple fruit employing LC/MS. Spot 6126 identified from 2D gel (a)
was excised and digested with trypsine and corresponding peptides were analyzed on a LC/MS system equipped with a nanospray ion source. Total
ion chromatography is shown in (b). During LC/MS analysis, two peptides were detected and their MS/MS spectra were collected at retention time
of 24.45 and 29.60 min, respectively (c) and (d). The MS/MS ion search employing MASCOT revealed doubly charged GAEILEGNGGPGTIK and
IFTGEGSQYGYVK, respectively as peptide 1 and 2. The spot no. 6126 was therefore identified as major allergen mal d1 protein (MW: 17,69 kDa, pI
5.67). More detailed information can be obtained. (From Zheng, Q. et al. 2007. J. Agric. Food Chem. 55:1663–1673.)
Advances in Postharvest Fruit and Vegetable Technology
Recent Developments in Proteomic Analysis of Fruits 317

using heavy versus light isotope coded tags in two comparable samples. The relative
abundance of proteins can be analyzed as peak-doubling in the same MS spectrum,
thus reducing the analytical noise, and increasing the quantitative information of
comparable sample data (Gygi et  al., 1999). Similarly, stable isotope labeling by
amino acids in cell culture (SILAC) allows differential metabolic labeling, followed
by mass analysis of cell culture samples (Ong et al., 2002). Each labeling technique
has its own advantages and disadvantages. For example, iCAT fails to quantify pro-
teins with no cysteine residues, and it can only compare two samples at a time. Unlike
iCAT, SILAC is an in vivo labeling method, which requires labeling on the amino
acids involved in the growth medium. Among the available labeling approaches,
another labeling on peptides, using amine reactive isobaric peptide derivatization
reagents, such as iTRAQ (isobaric tags for relative and absolute quantitation), has
been developed (Ross et  al., 2004). These derivatized peptides are indistinguish-
able by their MS spectra or MS/MS series, but exhibit intense, low mass MS/MS
signature ions (m/z 114–117), also called reporter ions, that permit quantitation of
each member of a multiplex set, where four comparable samples can be analyzed in
parallel (Ross et al., 2004). This iTRAQ approach can be applied to a set of samples
up to 8, with quantitative information on peptides. As a recent development, iTRAQ
has been applied on many plant research samples, such as Arapidopsis (Dunkley
et al., 2004; Zieske, 2006), and fruit proteomes in grapes (Luecker et al., 2009) and
tomatoes (Pan et al., 2014).
Another labeling strategy involves methylation of peptide amino groups via reduc-
tive amination with isotopically coded formaldehydes (Hsu et al., 2003). This tech-
nique was further developed by coupling it with 2D-LC-MS/MS (Boersema et al.,
2008; Boersema et al., 2009). In this method, all primary amines (the N-terminus
and the side chain of lysine residues) in a peptide mixture are converted to dimethyl-
amines. By using combination of several isotopomers of formaldehyde and cyanobo-
rohyride, peptide triplets can be obtained, that differ in mass by a minimum of 4 Da
between different samples. Extracted proteins are digested with protease (trypsine),
and the derived peptides are labeled with isotopomeric dimethyl labels, which are
then mixed, and simultaneously analyzed, by LC/MS. The quantitation is based on
the integration of the entire extracted ion peaks of each of the three m/z values for a
peptide triplet, and used to compare the peptide abundance in the difference s­ amples.
The modification provides enhanced confidence in protein abundance ratios, statis-
tical evaluation of significance, and discards proteins identified with only a single
peptide (Melanson et al., 2006). As a new technique, an isotopic dimethylation label-
ing technique was applied to apple fruit, to investigate the significantly changed
proteins during fruit ripening, and in response to ethylene treatment. This study lead
to the identification and quantitation of more than 1000 proteins in apples (Jun Song,
unpublished). Use of the technique to quantify apple mal d1 allergen protein during
apple fruit ripening, and in response to ethylene treatment, is shown in Figure 14.2.
Direct comparison of iCAT, iTRAQ, DIGE, and 2-DE based techniques showed
that DIGE had the same sensitivity as iCAT, but DIGE showed the shortcomings, such
as comigration and partial comigration of spots, which may lead to misidentification
of spots, and compromise the accuracy of quantitation of proteins (Wu et al., 2006).
The iTRAQ technique showed the highest sensitivity, but it can only applied on
318 Advances in Postharvest Fruit and Vegetable Technology

FIGURE 14.2  Example of quantitative proteomic analysis of apple proteins employing

dimethylation isotope labeling during fruit ripening and in response to ethylene treatment.
Among the identified and quantified 790 proteins, a major allergen, mal d1 (gi|4590376,
MW:17.679 kD), was identified and quantified, based on a total of 32 peptides at day 21,
after ethylene treatment at 20°C (Tables 14.1 and 14.2). The dimethylation resulted in a mass
shift of 0 per label (light) or 4 per label (medium) or 8 per label (heavy). Changes in doubly
charged peptide GAEILEGNGGPGTIK +2, one of the 32 quantified peptides, which has
two labeling sites (both N-terminus and K), changed significantly among the labels from
three different samples, control day 0, labeled as H (heavy, +8), ethylene treated at day
21, labeled as M (intermediate, +4) and control at day 21 as L (light). The total quantita-
tive change of mal d1 protein was summarized by 34 labeled peptides from three different
samples. The relative quantitative changes of mal d1 was shown to increase by 3.36 ± 1.94
folds during fruit ripening and further increased by 1.70 ± 1.28 folds to ethylene treatment.
(Jun Song, unpublished.)

certain types of mass spectrometry instruments. Nevertheless, all three methods are
complementary, and provide improved information for quantitative proteomics (Wu
et al., 2006). An investigation on comparison between 2D LC technique and 2D–
DIGE was conducted on rice proteome samples (Komatsu et al., 2006), and showed
that 2D LC resulted in better detection of low abundance proteins, but it showed

TABLE 14.1
Sequence Table of Quantitative Changes in Peptide GAEILEGNGGPGTIK
(an Apple Major Allergen Protein, Mal.d1) in Apples Employing Dimethylation
Isotope Labeling during Fruit Ripening and in Response to Ethylene Treatment
L/M SD L/H SD Fraction Correlation Intensity Modifications
17 GAEILEGNGGPGTIK 0.87 0.11 2.87 0.13 0.67 0.97 3222
Recent Developments in Proteomic Analysis of Fruits 319

TABLE 14.2
Quantitation Table of Some Allergen Proteins in Apples Employing
Dimethylation Isotope Labeling during Fruit Ripening and in Response to
Ethylene Treatment
Accession Sore Mass (Da) L/M SD # L/H SD # Description
1.1 gi|4590368 2296 18,106 0.58 1.61 41 3.73 2.08 41 Malus domestica
1.2 gi|2443824 1952 18,059 0.54 1.55 35 3.59 1.95 35 Malus domestica
1.3 gi|4590376 1598 18,208 0.59 1.26 32 3.36 1.94 32 Malus domestica

poor reproducibility and, therefore, required a large number of repeats, enough for
determining statistical significance. It also demonstrated that the 2D-DIGE method
is comparable to 2D LC for quantitative comparisons, with very good sensitivity
(Komatsu et  al., 2006). Techniques such as MudPIT are well suited for the iden-
tification of hydrophobic proteins (Link et  al., 1999; Washburn et  al., 2002), and
peptide sequencing by mass spectrometry (Steen and Mann, 2004). On the other
hand, various peptide labeling techniques, such as ICAT (Gygi et al., 1999), SILAC
(Ong et al., 2003) or iTRAQ (Ross et al., 2004), have become more frequently used
for proteomic research.
Proteins undergo a variety of post-translational modifications (PTMs) in a liv-
ing cell. Phosphorylation is one of the important PTMs of proteins, and plays an
important role in proteomic studies, especially involved in cellular signaling path-
ways in plants and fruit (Peck, 2006). A strategy that combines the application of
enrichments of both phosphorylated protein and peptides, with a separation of pro-
teins analysis, was reported for Arabidopsis and M. truncatula (Laugesen et  al.,
2006). Affinity chromatography with immobilized metal ions (IMAC) was shown to
allow the characterization of a high number of phosphorylation sites in Arabidopsis
(Nuehse et  al., 2007). In addition, three ethylene-enhanced and three ethylene-
repressed unique phospopeptides in Arabidopsis mutant ein2 were also identified
(Li et al., 2009). There have, however, not been any reports on PTMs of proteins in
fruit and vegetables.
Another MS-based proteomic approach based on the selection of proteolyitc
peptide detection as a targeted proteomic technique, has been developed (Anderson
and Hunter, 2006). In this approach, protein identification and quantitation can be
achieved by a selected reaction monitoring (MRM/SRM) technique, using triple
quadrupole mass spectrometer. Peptide mixtures are separated by liquid chroma-
tography, and then ionized, and detected. In the MRM mode, the mass spectrometer
can be programed to only the list of target proteotypic peptides. Only the predefined
precursor ions can be selected in the first quadrupole. A second mass filter, in the
third quadrupole, allows filtering of the corresponding fragment ions, following
collision-induced dissociation in the second quadrupole (Lange et al., 2008). The
resulting precursor fragment ion pairs (transitions) are highly specific for a given
proteotypic peptide, and are, therefore, unique for a given protein. By including
isotope-labeled versions of the proteotypic peptides that are being analyzed, it is
possible to simultaneously determine the absolute amounts of the protein. The
320 Advances in Postharvest Fruit and Vegetable Technology

approximate retention-time information can be used to restrict the time for the
detection of a specific transition, and, therefore, allows the detection of multiple
peptide ions in one measurement, a process that is referred to as scheduled selected
reaction monitoring.
The detection range can be extended to single-digit copies/cell and to proteins
undetected by classical methods (Picotti et al., 2010). Recently, a proteomic proto-
col combining OFFGEL and MRM studies to identify and quantitatively investigate
monodehydro-ascorbate reductase, a key enzyme in apple fruit during fruit ripening,
was reported (Yang et al., 2014).

14.3.3 Informatics/Bioinformatics
Mass spectrometry is the central technology for proteomic research, but several
approaches can be applied for peptide and protein identification. For example, a set
of peptide molecular masses from a specific enzyme digested proteins, peptide mass
fingerprint (PMF), can be generated and determined, using a matrix-assisted laser
desorption ionization (MALDI) MS. The experimental spectrum is compared with
theoretical ones computed from protein sequence databases to create “similarity
scores” for candidate protein (Henzel et al., 1993; Mann et al., 1993). PMF offers a
fast and simple analysis, but it can only be used with a pure protein or a simple mix-
ture, such as a spot from the 2-D gel (Palagi et al., 2006). Using tandem mass spec-
trometry (MS/MS) analysis, ionized peptides are isolated and fragmented (Pandey
and Mann, 2004). During the analysis, the peptide is analyzed at the first stage of
the mass analysis, then induced to fragment by collision, to generate a second mass
analysis. The collected MS/MS spectra will then be used to match proteins in a
database. Another limitation is that a database of protein or nucleic acid sequence is
required, and it is therefore not applicable to an unsequenced fruit species.
One of the most applied common strategies in the “proteomic pipeline” is called
MS/MS ion search. It uses software to search the uninterpreted MS/MS data from
a single peptide, or an LC/MS run, to match the peak list. A peptide sequence is
matched to individual MS/MS spectrum by a cross correlation algorithm, to com-
pare experimental MS/MS spectra against predicted peptide sequence from a data-
base (Eng et al., 1994). It should be remembered that it is peptide being identified,
not the protein. To ensure the accuracy or minimize the false positive, two levels of
control become necessary. First, the match of a peptide to a database must be esti-
mated by determining the false positive rate (FDR). Using identical search param-
eters, the search can be repeated against a database, in which the sequence have been
reversed or shuffled. Another level of control is to exam the identified protein family.
Depending on the rules for protein and the protein family, it is usually advisable to
use at least two identified peptides to call significant match of a protein.
Statistical analysis in comparative proteomics is one of the most important data
analysis processes involving numerous variables, many of which cannot be con-
trolled. Experiment design, biological repeats, and technical replicates, are necessary
to ensure the validation of any proteomic study. This implies that data normalization
and statistical analysis must be optimized for accurate detection of differentially
expressed proteins. A review on design and analysis issues in quantitative proteomics
Recent Developments in Proteomic Analysis of Fruits 321

studies was provided by Karp and Lilley (Karp and Lilley, 2007). Clear guidelines
for publication of proteomic research data, which included statistical analysis, bio-
logical design, and normalization research results, should be followed (Carr et al.,
2004; Valledor and Jorrín, 2011; Wilkins et al., 2006).

14.4  PROTEOMIC RESEARCH ON APPLE AND PEAR

Although the genome sequences of most fruits are incomplete, numerous proteomic
studies have been conducted to reveal the key proteins in relation to metabolic path-
ways and specific functions in many fruit (Palma et  al., 2011). Several proteomic
investigations have been carried out on apple (Buts et  al., 2014; Guarino et  al.,
2007; Qin et al., 2009; Zheng et al., 2007; Zheng et al., 2013) and pears (Pedreschi
et al., 2007, 2008). Most proteomic techniques have used 2-DE technology, using
IPG strips, IEF/SDS-PAGE, DIGE and MS. Recent publications on apple (Malus
domestica) and pear (Pyrus pyrifolia) fruit proteomic research are summarized in
Table 14.3.
Early proteomic work conducted on apples, identified a few proteins in relation
to fruit-ripening and as well as some glycoproteins. A proteomic investigation con-
ducted by Guarino et  al. (2007) on apple (Malus domestica cv. “Annurca”) flesh
tissue, using 2-DE and MALDI-TOF as well as LC-MS/MS, identified 44 pro-
teins, which were significantly involved in energy production, ripening, and stress
responses, but also some proteins as fruit allergens. (Guarino et al., 2007) Another

TABLE 14.3
Publications on Proteomic Research with Apples and Pears
Plant Proteomic No. Proteins
Material Target Technique Identified Reference
Apple Sample 2-DE, MTa, and 32 Guarino et al. (2007)
(Malus preparation LC-MS/MS
domestica) Fruit ripening 2-DE, LC-MS/MS 3, 13 Guarino et al. (2007), Zheng
OFFGEL-LC/MS/ 1709 et al. (2013), Yang et al. (2014)
MS, MRM
Fruit senescence 2-DE, LC-MS/MS 34, 189 Qin et al. (2009), Zheng
(2013)
Fruit senescence Label free (DID) 287b Buts et al. (2014)
Allergen 2-DE, LC-MS/MS 5, 4 Song et al. (2006), Wu et al.
(2008), Zheng et al. (2007,
2013)
Herndl et al. (2007)
Pear (Pyrus Disorder 2-DE, LC/MS/MS 1 Carpentier et al. (2005)
communis) Fruit ripening 2-DE, MT, and 43, 22, 151 Pedreschi et al. (2007, 2009)
LC-MS/MS Pedreschi et al. (2008)

a MT: MALDI-TOF.
b Protein families with 2528 peptides.
322 Advances in Postharvest Fruit and Vegetable Technology

proteomic study on “Red Delicious” apple fruit, using 2-DE, identified three aller-
gens in fruit peel samples (Zheng et al., 2007). In other study from the water-soluble
fraction of an apple extract, four previously classified allergens, Mal d1, Mal d2, Mal
d3, and Mal d4, were identified in Western blots with polyclonal rabbit antibodies
directed toward the four respective allergens. All four known apple allergens were
localized on a 2-DE map, and they were matched with spots recognized by sera of
patients with different allergic patterns, whilst a new, putative allergen, was also
identified using MS (Herndl et al., 2007).
Apple fruit senescence at the proteomic level was also investigated, using com-
parative proteomic techniques to characterize the dynamic alterations in the mito-
chondrial proteome during fruit senescence. In purified mitochondria from apple
fruit under various stages of senescence, Qin et al. (2009) identified a set of 22 pro-
teins spots that appeared to change significantly in abundance. These proteins are
mainly involved in metabolism related to TCA cycle, respiratory chain, and carbon
metabolism. They also showed that alteration of energy metabolism contributed
to cellular and organismic senescence (Qin et  al., 2009). A proteomic approach,
employing a 2-DE technique with SYPRO Ruby, was performed by Zheng et  al.
(2007) to separate the total proteins from apple fruit at different stages of ripening,
and senescence, after ethylene treatment. From 2340 spots, a total of 316 spots, or
approximately 14% of the total protein population, was found to be significantly
changed. Of these, 219 spots were only present at a specific ripening stage, while
97 spots varied throughout ripening, and in response to ethylene treatment. From
the 316 candidate spots, 221 proteins were further identified by liquid chromatog-
raphy and MS, with protein sequence, and express sequence tag database search.
This indicated that apple fruit ripening is associated with an increased abundance
of proteins, with functions such as ethylene production, antioxidation and redox,
carbohydrate metabolism, oxidative stress, energy, and defense response. Ethylene
treatment increased a number of unique proteins that were not present during normal
fruit ripening, and reduced some proteins involved in primary metabolism, includ-
ing those of the last few steps of the glycolytic pathway. This study demonstrated the
complexity and dynamic changes of protein profiles of apple fruit during ripening,
and in response to exogenous ethylene treatment.
Using 2-DE and LC-MS/MS, Pedreschi et al. (2007) identified proteins related
to brown core, a physiological disorder in “Conference” pear fruit during storage.
Seventy-eight proteins were identified as being related to the development of this
disorder. After the protein profiles from each group were analyzed, the upregulated
proteins in brown tissues were found mainly involved in energy and defense systems.
In this study, a unique approach, using univariate and multivariate statistical tech-
niques, seems to be an efficient method to elucidate the mechanisms and pathways
leading to the disorder. Another study conducted by Pedreschi et al. (2008) inves-
tigated the combination of oxygen and carbon dioxide concentrations, precooling
period, and harvest maturities, on core breakdown, presumably in the protein levels
during controlled atmosphere storage of “Conference” pears. The results demon-
strated that impaired respiration is highly related to protein synthesis alterations,
and the activation of defense mechanisms. Triosephosphate isomerase, a key enzyme
of the energy metabolism, was shown to be upregulated under browning-inducing
Recent Developments in Proteomic Analysis of Fruits 323

conditions, in an attempt to use alternative, more efficient, anaplerotic pathways, to

cope with the applied stresses. The changes in the accumulation of proteins related
to ethylene biosynthesis (ACC oxidase) and allergens (major allergen Pyrc 1) were
highly dependent on the oxygen and carbon dioxide concentrations. ACC oxidase
and Pyrc 1 were also clearly downregulated under low oxygen, or high carbon diox-
ide, concentrations (Pedreschi et al., 2008).
In addition, a new proteomic study, applying label-free technique, was developed,
using data-independent acquisition to investigate the apple proteome change during
the postharvest period. It identified 287 proteins families in “Braeburn” apples that
increased significantly after 120 days of storage (Buts et al., 2014).
Despite limited numbers of publications and identified proteins, these examples
clearly indicate that comparative proteomics, combined with various MS technolo-
gies and bioinformatic tools, are powerful and important tools to reveal numerous
physiological and biological processes during ripening and development, as well
as to reveal physiological disorders in fruits and vegetables. Data from apple and
pear research demonstrated that proteomic tools provide an additional tool, besides
genomics, to research fruit allergens.

14.5  FUTURE RESEARCH PERSPECTIVES

Despite the significant advances in technology, proteomic research on fruit and
vegetables has been limited mainly to gel-based 2-DE systems, with MS protein
identification. In order to improve the coverage and quantitative protein analysis,
nongel-based and high throughput techniques should be applied to study complex
fruit proteomes. Efficient sample preparation into fractions and cost-effective new
labeling techniques also need to be developed, and applied to fruit proteomics
research. New analytical concepts, such as targeted proteomics (MRM/SRM), are
being explored, to overcome many of the existing limitations, and MS instrumenta-
tion will continue to improve the sensitivity and accuracy of current MS measure-
ments. It will be equally important to develop an effective computational framework
for the integration of proteomics data with phenomic and functional genomic infor-
mation, to reconstruct molecular networks and how they interact in cells—an essen-
tial step in bridging the genotype–phenotype gap. Given the pivotal role that proteins
play in fruit physiology, proteomic techniques, such as high throughput studies on
protein abundance and activity, become very useful and necessary. Protein-based
microarrays offer the possibility to investigate the specific proteins, using antibodies
to conduct high throughput studies, by printing a collection of target proteins on the
array surface, and assess their interactions and biochemical activities (Labaer and
Ramachandran, 2005). Immunolocalization, immunoblot, and fluorescence imaging
techniques, could also help to determine protein expression, localization, activity
state, and cell compartmentation (Giepmans et al., 2006).
One important aspect is the lack of proteomic studies on protein PTMs during
postharvest ripening and handling. PTMs play an important role in cell regulation
and signal transduction. MS is the tool of choice for the analysis of protein PTMs,
but without prior knowledge, PTMs can be profiled and quantified at the amino acid
level, and used to elucidate a signal transduction pathway.
324 Advances in Postharvest Fruit and Vegetable Technology

Another important future research direction of fruit proteomics is to study protein

complexes, and their interaction. Investigations on those protein complexes, such as
interactions of protein–protein, protein–DNA and protein–RNA, will provide fur-
ther insights into fruit-ripening initiation, and regulation. As a common practice,
the immunoprecipitation techniques have been developed and applied to protein
interactions (McHugh et al., 2014; Thelen and Miernyk, 2012). Ethylene-regulated
climacteric ripening, as well as the response of fruit to environment stresses, pro-
vide long-term, intriguing biological questions for postharvest physiologists who can
apply state-of-the-art proteomic research tools to address these challenges. With
the further development of proteomic research tools/platforms, including bioinfor-
matics, it is certain that proteomics will provide unique and important insights into
the regulation of fruit-ripening, an important sector of systems biology, and future
marker selection, and validation for breeding programs.
Although few of the genomes of fruits and vegetables have been completely
sequenced so far, many genomic tools available for Arapidopsis (Arabidopsis
Information Resource), Solanaceae (Solanaceae Genomic Network), and Rosaceae
(Rosaceae Genome Database) can be applied to future research and development
in proteomics (Wheeler et al., 2005). In comparison, apple has more than 60,000
protein sequences in NCBI (accessed on December 2014). A high quality draft of
the complete genome of pear (Pyrus × bretschneideri), reporting 42,812 protein
coding genes, and with ca. 28% encoding multiple isomers (Wu et al., 2013), there
were 46,518 protein sequences for public use on http://www.ncbi.nlm.nih.gov/pro-
tein (accessed on December 2014). Undoubtedly, the ultimate success of proteomic
research is based on the completion of the genome sequences of fruits and vegetables.

ACKNOWLEDGMENTS
The author thanks Drs. G. Braun at AAFC, J. Golding, and R. Wills, for their critical
reviews and constructive suggestions in preparing this manuscript.

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 15 Organic Postharvest
Technology
Apiradee Uthairatanakij and Pongphen Jitareerat

CONTENTS
15.1 Introduction................................................................................................... 331
15.1.1 What Are Organic Fruits and Vegetables?........................................ 332
15.2 Postharvest Handling Systems for Organic Produce..................................... 332
15.2.1 Handling at Harvest........................................................................... 332
15.2.2 Waxing............................................................................................... 333
15.2.3 Storage............................................................................................... 334
15.2.4 Transportation and Distribution........................................................ 334
15.3 Disinfection of Organic Produce................................................................... 334
15.3.1 Chlorine............................................................................................. 335
15.3.2 Ozone................................................................................................. 336
15.3.3 Peroxyacetic Acid.............................................................................. 336
15.3.4 Organic Acids.................................................................................... 336
15.3.5 Electrolyzed Water............................................................................. 337
15.3.6 Hydrogen Peroxide............................................................................ 337
15.4 Postharvest Treatments for Organic Produce................................................ 337
15.4.1 Modified Atmosphere Packaging...................................................... 337
15.4.2 Heat Treatment.................................................................................. 337
15.4.3 Antibrowning Agents for Fresh-Cut Produce.................................... 338
15.4.3.1 Ascorbic Acid..................................................................... 338
15.4.3.2 Carboxylic Acids................................................................. 339
15.4.3.3 Ultraviolet Radiation........................................................... 339
15.4.3.4 Ultrasound...........................................................................340
15.5 Postharvest Quality of Organic Produce.......................................................340
15.6 Conclusions....................................................................................................340
Acknowledgment.................................................................................................... 342
References............................................................................................................... 342

15.1 INTRODUCTION
Organic agriculture has a number of potential environmental benefits and is con-
sidered a positive force for green consumerism. These potential benefits have led to
increased production and consumption of organic produce; this is one of the major
market trends of this decade. To ensure this success, organic fruits and vegetables

331
332 Advances in Postharvest Fruit and Vegetable Technology

at harvest are expected to be of high quality in terms of color, shape, texture, and
unique flavor, and must be free from decay and injury, as expected of conventionally
grown produce. In addition, the postharvest handling system for organically grown
produce must follow the guidelines for organic commodities, and have the necessary
documentation to demonstrate compliance with these standards from the farm to the
consumer.

15.1.1 What Are Organic Fruits and Vegetables?

The term “organic” refers to the way that agricultural products are grown, handled,
and processed. Organic fruits and vegetables are produced using an organic farm-
ing system that emphasizes renewable resources and conservation of soil fertil-
ity and water, rich biodiversity, and long-term sustainability (Plotto and Narciso,
2006). This production system relies on crop-rotation, soil-building, and biological
pest management (UNCTAD, 2003). Similar production practices are also used for
conventionally grown produce, but organic production operates without the use of
synthetic fertilizers and pesticides. Postharvest, organically grown fresh fruits and
vegetables must be handled without synthetic chemicals, except for those specifically
allowed by regulation, and without ionizing irradiation. If these organic practices are
followed and certified, the products can be labeled as “organic.”
Labeling is essential to allow consumers to recognize organic products and dis-
tinguish them from conventional grown commodities (Hemmerling et  al., 2013).
Organic product labels are useful instruments to establish consumer trust and to
quickly allow consumers to identify organic commodities. At the same time, labels
help to avoid confusion and increase the profile of organic food, positively influenc-
ing the consumer’s choice of produce (Padel and Midmore, 2005).

15.2 POSTHARVEST HANDLING SYSTEMS FOR ORGANIC PRODUCE

The use of postharvest technology for organic produce, in order to achieve similar
outcomes as conventional crops, is one of the most challenging goals for organic
producers, handlers, and processors. An integrated approach for postharvest han-
dling is needed, one that incorporates “layers of prevention” to reduce chemical and
microbial contamination of fresh organic produce, but still maintains produce qual-
ity during handling, packing, storage, and shipping operations. A basic requirement
for certified organic produce is that there should no cross-contamination between
organic produce and prohibited substances. A major problem for shippers and han-
dlers is the need to segregate organic products from conventionally grown fruits and
vegetables, to avoid comingling. Ingredients and materials used postharvest must
also be organic or otherwise approved (Plotto and Narciso, 2006).

15.2.1 Handling at Harvest
Careful investigation is needed of all the steps in the postharvest chain, to develop
and refine a proper handling protocol that ensures that organic products are correctly
handled, according to organic protocols. In organic operations, the International
Organic Postharvest Technology 333

Federation of Organic Agriculture Movements (IFOAM, 2012) requires that han-

dlers shall not comingle organic produce with nonorganic produce throughout the
entire postharvest handling process.
The receiving area should be clean and free from prohibited substances such as
pest control products and other debris that may result in contamination of organic
produce or its comingling with nonorganic produce. Adequate sanitation and water
disinfection during postharvest handling are vital components of a postharvest man-
agement plan. Food safety issues are of concern with all horticultural crops, and
have become increasingly important to the sales and marketing of fresh fruits and
vegetables. Several cases of food-borne illnesses, due to contamination with human
pathogens, have been traced to improper sanitation during postharvest handling.
For example, organic sprouts from a farm in Illinois infected at least 140 people in
26 states in the United States with salmonella in 2012. In 2011, a massive outbreak
of a deadly strain of E. coli was linked to sprouts from an organic farm in Germany
that killed 50 people and sickened more than 4,300. However studies have shown no
significant difference in prevalence of E. coli and other pathogens between organi-
cally and conventionally grown produce (Mukherjee et al., 2004; Winter and Davis,
2006; Leifert et al., 2008; Oliveira et al., 2010). It is also estimated that 30% of fresh
produce is lost to microbial spoilage due to nonhuman pathogens and saprophytes
from harvest, through postharvest handling, and delivery to consumers. For organic
produce, disinfectants and other sanitation products must comply with specific
regulations (IFOAM, 2012; OMRI [Organic Materials Review Institute], 2013). If
unloading organic produce from bins involves floatation, only approved substances
may be used in the floatation water to reduce damage and cross-contamination
(Suslow, 2000). Allowed substances that include lignin sulfonates can be used in
certified organic handling (OMRI, 2013). For organic produce, packaging materials
such as storage containers, bins, and boxes cannot contain synthetic fungicides, pre-
servatives, fumigants, or nano-materials. Polyvinyl chloride (PVC) and aluminum
should be avoided.

15.2.2 Waxing
Water loss of organic fresh produce is a primary factor in loss of postharvest quality
and of saleable weight. Waxing or coating can be effective in reducing water-loss
in produce and can also enhance appearance. According to the National Organic
Program (NOP) in the USA, wax for organic produce must not contain any prohib-
ited synthetic substances, preservatives or fungicides, and may not have petroleum-
based ingredients. Beeswax, wood rosin (resin from pines), and carnauba (from
palm leaves) are allowed (www.ams.usda.gov/standard/). Natural waxes and resins
are difficult to apply directly to fruit and vegetables as coating, without first mak-
ing an emulsion. Emulsification of natural waxes and resins for fruit and vegetable
coatings are made by adding fatty acids at high temperatures (Plotto and Narciso,
2006). Commercial carnauba wax-coating often contains a fatty acid (10%–30% of
solids), ammonia or morpholine (3%–10% of solids), and antifoam (Hagenmaier and
Baker 1997). However, morpholine and ammonia are prohibited for organic pro-
duce. Therefore, beeswax, wood rosin, and carnauba are emulsified with vegetable
334 Advances in Postharvest Fruit and Vegetable Technology

oil, vegetable-based fatty acids, and ethyl alcohol in water (www.ams.usda.gov/­

standard/). Only beeswax (INS 901) and carnauba (INS 903) are allowed in posthar-
vest handling by IFOAM regulations (IFOAM, 2012).

15.2.3 Storage
Temperature and humidity management inside the storage room play a key role in
determining the storage-life and shelf-life of organic produce, similar to conventional
produce. Proper cooling reduces the respiration rate and maintains visual quality,
texture, flavor and nutritional composition. Whatever the cooling method utilized,
if both organic and conventional produce are being handled, the organic produce
must be cooled at the beginning of daily operation to prevent cross-­contamination of
organic produce with prohibited substances. An acceptable practice is the injection
of ozone into the cooling water to reduce pesticide residues that may remain in the
water after cooling nonorganic produce (Suslow, 2000). In addition, the same restric-
tions on the storage of conventionally produced commodities, especially those from
the tropics and subtropics sensitive to chilling injuries, applies to organic produce.
Ethylene is allowed for postharvest ripening of tropical fruits and degreening of
citrus fruits.

15.2.4 Transportation and Distribution

Organic produce must be segregated from nonorganic conventional produce to avoid
cross-contamination. Thus, organic produce should never be stacked beneath con-
ventional produce during storage and transport. When organic produce is being
shipped in the same truck, it must be packed in separate boxes and on separate pal-
lets to prevent cross-contamination with conventional produce and must be clearly
labeled as “organic.” A full review of the storage, transportation, and distribution
center is essential to identify potential risks for comingling. These reviews need to
be carried out on a regular basis and documented.
Upon shipment arrival, the organic produce cartons should be in good condition,
without rips and tears or damage. Broken packaging may mean that the organic
produce could have been cross-contaminated with nonorganic produce. Also, any
organic packages accepted during receiving should be clearly labeled as organic and
have a certifying agent’s name.
During storage at a distribution center, a barrier is required between packed and
unpacked conventional and organic produce, using space buffers. If repacking is
necessary, packing materials and storage containers for organic produce must be
free from synthetic fungicides and fumigants.

15.3  DISINFECTION OF ORGANIC PRODUCE

Microbial contamination in organic produce is a critical point for both shelf-life and
consumer safety. To achieve this goal, many organic and conventional growers have
implemented Good Agricultural Practices (GAPs) during production and harvest
operations to ensure quality and minimize contamination. Also, many packers or
Organic Postharvest Technology 335

handlers have implemented Best Management Practices (BMPs) to maintain quality

and reduce the microbial load on fresh organic produce after harvest.
The postharvest handling operation of organic produce cannot use unapproved
chemicals and most synthetic inputs are prohibited. Those synthetic inputs allowed can
only be used with restrictions. Additionally, there are no internationally recognized
regulatory agencies harmonizing the approval of either biopesticides or chemicals for
use in organic commodities, postharvest (Prange et al., 2006). The United States and
the European Union recently accepted each other’s organic standards as equivalent,
with minor exceptions in animal production. For other c­ ountries, chemicals used in
organic postharvest operations must comply with the organic ­requirements of each
country. Of particular interest are the types of sanitizing agents that can be used.

15.3.1 Chlorine
Chlorine is the most common disinfectant that can be added to transport flumes,
or to produce cooling, or in wash-water. In water, chlorine exists in equilibrium
as hypochlorous acid and hypochlorite ion, depending upon the pH. Hypochlorous
acid provides the strongest antimicrobial properties. At pH 6.5, 95% of the chlorine
is in  the hypochlorous form; therefore, maintaining the water pH at the range of
pH 6.5–7.5) provides the greatest disinfecting power. Chlorine gas will be released
if highly acidic water is used (Suslow, 2000). Products used for adjusting acidity
in water must be from natural sources such as citric acid, sodium bicarbonate, or
vinegar (Suslow, 2000). Chlorine in the water reacts with organic compounds to
produce chlorinated compounds suspected of detrimental effects on humans and
wildlife (Suslow, 2006). The most common compounds are trihalomethanes and
haloacetic acids.
Organic processors and shippers may use chlorine within specified limits. All
liquid sodium hypochlorite, granular calcium hypochlorite, and chlorine dioxide are
restricted materials by organic standards. The application of chlorine must conform
with the Maximum Residual Disinfectant Limit under the Safe Drinking Water Act
at 4 mg/L (ppm) expressed as chlorine (Suslow, 2006; Silva, 2008). Sodium hypo-
chlorite is typically used as a source of chlorine.
The chlorine levels in the wash-tanks and flumes need to be continuously moni-
tored. Chlorine readily binds to soil, debris, and other organic matters in the water,
and is no longer available for disinfection (Silva, 2008). A common way is to monitor
both the pH and the oxidation-reduction potential of the wash-water. The NOP stan-
dard permits a threshold of 4 ppm residual chlorine in the effluent. From handling
organic products, the downstream product-wash needs to be monitored to ensure that
the effluent water does not exceed this limit. California Certified Organic Farmers
(CCOF) has recently modified this threshold to permit 10 ppm residual chlorine
measured downstream of the wash. However, the levels of chlorine used to prepare
water for sanitation of equipment, tools, product surfaces, or edible products, should
be in high enough concentrations to control microbial contaminants. In order to
maximize the antimicrobial activity, it is beneficial to prewash the produce arriv-
ing from the field, or before loading. This may include a vigorous prewash with
brushes or sponges to remove excess debris from the produce, or a clear-water rinse
336 Advances in Postharvest Fruit and Vegetable Technology

to remove soil and other debris, prior to using the sanitizer solution. Also, cleaning
dump tanks and residue screens helps to minimize the presence of soil and debris,
and maximize chlorine effectiveness.

15.3.2 Ozone
Ozone is considered to be GRAS (Generally Regarded as Safe) for fresh produce and
equipment, and ozonated water is becoming an increasingly popular effective alter-
native to chlorine for postharvest application, due to it not producing any unaccept-
able byproducts, and it has a higher antimicrobial activity than chlorine (Kim et al.,
2003; Ölmez and Särkka-Tirkkonen, 2008). Ozone provides comparable disinfec-
tion power to chlorine, and, in addition, attacks bacterial cell-walls and thick-walled
spores of plant pathogens. Ölmez and Kretzschmar (2009) stated that low concentra-
tions (1–5 ppm) of ozone at short exposure times (1–5 min) is effective against many
bacteria, yeasts and molds, as well as some viruses, but it has low stability of less
than 20 min in clean water and must be generated continuously in situ (Silva, 2008).
A high level of ozone can lead to produce injury such as damage to the surface of
lettuce (Kim et al., 2006), and browning of iceberg lettuce (Koseki and Isobe, 2006).
An added advantage of ozone treatment is that its activity is not dependent on pH,
as chlorine is. It is therefore not necessary to adjust the wash-water pH (Ölmez and
Särkka-Tirkkonen, 2008). However, the efficacy of ozone treatment for disinfection
is depending on several factors, including types of microorganism, fresh produce,
the level of initial inoculum, the growth stage of microorganism, and the application
method of the ozone treatments (Ölmez and Kretzschmar, 2009). In addition, the
threshold level of ozone for a safe working environment are: 0.1 for long period (8 h);
and and 0.3 ppm for short period (15 min) (Ölmez and Särkka-Tirkkonen, 2008).

15.3.3 Peroxyacetic Acid
Peroxyacetic acid (PAA), also called peracetic acid, is allowed in an organic post-
harvest system. The disinfection performance of PAA is comparable to chlorine and
ozone in eliminating microbial biofilm in dump-tank and flume sanitation. Like
ozone, the treatment results in safer byproducts than chlorine. However, a disad-
vantage of PAA is its higher unit cost. To maximize effectiveness, PAA should be
maintained at a level of 80 ppm in the wash-water, and clean water is required for
washing after a PAA disinfection treatment (Silva, 2008).

15.3.4 Organic Acids
Many organic acids, such as citric acid, lactic acid, acetic acid, and ascorbic acid, have
been applied to minimize microbial growth in fresh and fresh-cut produce due to their
low pH of 2.1–2.7 (Arites et al., 2009; Ölmez and Kretzschmar, 2009). Their antimi-
crobial efficacy is greater on bacteria than yeasts and molds (Arites et al., 2009). The
efficacy of these organic acids against microbes is dependent on the type and concen-
tration of organic acid, water quality (such as pH, temperature, turbidity, and organic
content) used to dissolve the organic acid, application method (dipping, spraying),
Organic Postharvest Technology 337

exposure time (generally between 5 and 15 min), and target microorganisms, and inoc-
ulum level (Gil et al., 2009; Ölmez and Kretzchmar, 2009; Alexopoulos et al., 2013).

15.3.5 Electrolyzed Water
Electrolyzed water (EW) is an alternative disinfectant of fresh and fresh-cut pro-
duce, as it is safer and more ecologically friendly than chlorination. EW is gener-
ated by electrolysis of small amount of sodium chloride (0.1%–1.0%) in wash-water
(Arites et al., 2009; Ramos et al., 2013). Two types of EW have been used as the sani-
tized water: neutral electrolyzed water (NEW) and acidic electrolyzed water (AEW).
Both types strongly inactivate spoilage microorganisms and food-borne pathogens
(Ramos et  al., 2013). They are more effective than hypochlorite due to their high
oxidation–reduction potential (Izumi, 1999).

15.3.6 Hydrogen Peroxide
Hydrogen peroxide (H2O2) is used as a disinfecting agent due to its strong oxidiz-
ing power, and generates phytotoxic hydroxyl free radicles. Hydrogen peroxide is
allowed in food processing and packaging as it does not leave harmful residues
and decomposes into water and oxygen through the action of catalase (Arites et al.,
2009; Ölmez and Kretzchmar, 2009). The concentration of hydrogen peroxide used
in wash-water as the antimicrobial agent ranges between 0.04 and 1.25% (Ölmez and
Kretzchmar, 2009). However, Ramos et al. (2013) recommend a higher concentration
of 2%–4%, though concentrations of 4%–5% may be phytotoxic to fresh produce.

15.4  POSTHARVEST TREATMENTS FOR ORGANIC PRODUCE

15.4.1 Modified Atmosphere Packaging
Modified atmosphere packaging (MAP) is a method of extending the shelf-life of
both organic and conventionally grown fresh produce and is used widely for fresh-
cut produce. The technology involves the replacement of air inside the package
with a beneficial mixture of elevated carbon dioxide, and reduced oxygen, which
is achieved by the natural interplay between the respiration of the produce and the
transfer of gases through the packing material. The efficacy of MAP on extending
shelf-life is dependent on several factors, such as the type of produce, gas mixture,
storage temperature, packing material and hygiene during handling (Fonseca et al.,
2002; Sandhya, 2010). Since there is no addition of nonnatural materials in the sys-
tem, MAP is an acceptable technology for organic produce; however, the use of
MAP on specific organic produce has similar requirements and performance as for
conventional produce.

15.4.2 Heat Treatment
Heat treatment is an acceptable technology for organic produce, as it leaves no chemi-
cal residues, and has been approved as an insect quarantine treatment by the United
338 Advances in Postharvest Fruit and Vegetable Technology

States Department of Agriculture (USDA) Animal and Plant Health Inspection Service
(APHIS) against several pests (USDA, 1993), and is widely used for disease control. It
is considered as a safe alternative physical treatment to reduce the quantities of post-
harvest chemicals used to inhibit growth of pathogens and insects (Lu et al., 2010).
Hot-water treatments, by themselves, or in combination with other heat and
mechanical treatments, can substantially control postharvest diseases of fresh pro-
duce. Porat et  al. (2000) found that hot-water brushing (HWB) at 56°C for 20 s
reduced decay in organically grown citrus cultivars such as “Minneola” tangerines,
“Shamouti” oranges, and “Star Ruby” red grapefruit, but did not cause surface dam-
age, and did not affect fruit weight-loss or internal quality. The treatment of peaches
and nectarines with a drench of heated water at 55°C or 60°C for 20 s over rotating
brushes effectively controlled brown rot (Karabulut et al., 2002). Lydakis and Aked
(2003) concluded that vapor-heat treatments at 52.5°C or 55°C for up to 24 min.
can be applied to “Sultanina” table grapes to control grey mold disease, without
compromising fruit quality. Similarly, Lu et al. (2010) showed that heat treatment of
plums and nectarines delayed ripening (higher TA and TSS values), but did not have
an effect on lycopene synthesis. Immersion in water at 60°C for 60 s was effective
to reduce the incidence of brown rot (Karabulut et al., 2010). In addition, hot water
at 55°C for 5 min. significantly reduced postharvest decay in hybrid organic melons,
without affecting other qualities of the melons (total soluble solids, fruit firmness or
the color of mesocarp tissue) during storage at 13°C, and also showed lowest fresh-
weight-loss (Uthairatanakij et al., 2011).
In contrast, disinfestation procedures for mangoes and papaya by hot forced air
for 4 h at 50°C, led to faster softening and, occasionally, hard lumps in the flesh
after treatment (Shellie and Mangan, 1993). Heating ‘Hujin’ peaches by moist air at
37°C for 12 h is the most effective treatment to maintaining hardness and reducing
water loss, while hot water caused heat injury (Zhou et al., 2002). Fruit sensitivity to
heat treatments is modified by preharvest weather conditions, cultivar, time of heat-
ing, and subsequent storage conditions, and is related to the level of heat protective
proteins at harvest and the postharvest production of heat-shock proteins (Paull and
Chen, 2000).

15.4.3  Antibrowning Agents for Fresh-Cut Produce

There is a growing demand for minimally processed, fresh-cut or ready-to-eat fruit
and vegetables. The operational processes used to generate fresh-cut produce cause
cell damage that limits the subsequent shelf-life (Soliva-Fortuny and Martin-Belloso,
2003). A major issue is browning on the cut surfaces. Browning is due to the enzy-
matic oxidation of phenolic compounds by polyphenol oxidase (PPO) to o-quinones
and subsequent polymerization to brown tissue (Mayer, 1987). A wide range of anti-
browning agents have been developed, but their use on fresh-cut organic fruits and
vegetables is restricted to agents of organic origin or from natural sources.

15.4.3.1  Ascorbic Acid

Ascorbic acid (AA), recognized as a GRAS substance, is allowed in organic produce
(OMRI, 2013) to prevent browning. Its effectiveness arises from its ability to reduce
Organic Postharvest Technology 339

o-quinones back to their phenolic substrates (Hsu et al., 1988). Dips of AA and its
derivatives have been widely used in the concentration ranges of 0.5%–4%, and
have been applied in various fresh-cut produce (Soliva-Fortuny and Martin-Belloso,
2003). Generally, AA is applied in combination with organic acids such as citric acid
(Pizzocaro et  al., 1993) and a mixture of 1% AA + 0.2% citric acid inhibits PPO
90%–100% in apple cubes. Moreover, the combination of 1% AA + 0.5% CaCl2 pre-
serves the color of apple cubes under appropriate MAP conditions (Soliva-Fortuny
et al., 2001, 2002). However, this treatment is not completely effective in controlling
enzymatic browning of fresh-cut fruit, since once the AA is completely oxidized to
dehydroascorbic acid, o-quinones are no longer reduced, and browning may occur
(Nicolas et  al., 1994). In addition, AA may cause important oxidative damage in
fresh-cut “Fuji” apples (Larrigaudiere et al., 2008).

15.4.3.2  Carboxylic Acids

Carboxylic acids have been widely used commercially due to their antibrowning
activity. Citric acid exerts a double inhibitory effect by reducing pH and chelating
copper in the active site of PPO and, thus, inactivating the enzyme (Son et al., 2001).
Optimum PPO activity is observed at pH 6.0–6.5, while little activity is detected
below pH 4.5 (Whitaker, 1995). Pizzocaro et al. (1993) reported more than a 90%
inhibition of PPO activity in apple cubes by using a mixture of 1% AA + 0.2% citric
acid or 1% AA + 0.5% sodium chloride. Other carboxylic acids, such as oxalic acid
and oxaloacetic acid, show higher antibrowning activity than citric acid on fresh-cut
apples (Son et al., 2001). Immersion of banana and apple slices in oxalic acid solutions
is effective in reducing browning (Son et al., 2001; Yoruk et al., 2004). Although the
mechanism of browning inhibition is unknown, oxalic acid seems to inhibit PPO per
se, by chelating copper from the active site of the enzyme, and has a high affinity to
copper ions forming a copper II metal complex (Tong et al., 1995). The extent of inhi-
bition is influenced not only by oxalic acid concentration, but also by pH (Altunkaya
and Gokmen, 2008). In addition, PPO enzymes from different sources exhibit differ-
ent types of inhibition mechanisms (Son et al., 2001; Aydemir and Akkanli, 2006).

15.4.3.3  Ultraviolet Radiation

Ultraviolet (UV) radiation can be divided into four types on the basis of wavelengths:
UV-A (400–320 nm), UV-B (320–280 nm), UV-C (280–200 nm), and UV vacuum
(Blaustein and Searle, 2013). Of these, UV-C has a high potential to inhibit microbial
growth in air, water, container surfaces, and vegetables, by the formation of pho-
toproducts in the DNA, called pyrimidine dimers. Pyrimidine dimer molecules are
generated on the same strand of DNA and are able to interfere with DNA transcrip-
tion and translation, leading to cell malfunction and cell dead (Franz et  al., 2009;
Bermudez-Aguirre and Barbosa-Canovas, 2013). The maximum antimicrobial activ-
ity of UV-C wavelengths is at 254 nm (Chang et al., 1985). UV-C is nonthermal treat-
ment for inactivation of microbial in food industry. The advantage of this technology
is safety, and leaving no chemical residue on wash-water and the produce. However,
the efficacy of UV-C for water disinfection is dependent on many factors, such as
turbidity, suspended solids, and the presence of absorbing compounds (Selma et al.,
2008). Graca et  al. (2013) showed that UV-C irradiation at 10 kJ/m2 could be an
340 Advances in Postharvest Fruit and Vegetable Technology

alternative treatment to washing with hypochlorite solutions for microbial disinfec-

tion of minimally processed apples. However, the use of UV-C treatment in the food
processing industry is limited, due to UV-C irradiation having multiple effects on
human skin, such as the risk of the common skin cancers, malignant melanoma, squa-
mous cell carcinoma, and basal cell carcinoma (Moon et al., 2005).

15.4.3.4 Ultrasound
Decontamination of food products can be achieved with nonthermal technology,
such as ultrasound. It has been extensively used to eliminate spoilage and food-borne
pathogens in a range of food products (Birmpa et  al., 2013). Ultrasound technol-
ogy is safe, nontoxic, and friendly to the environment (Feng and Yang, 2011). High
frequency of ultrasonic wave (≥20 kHz) leads to the chemical and physical changes
in biological structure (Butz and Tauscher, 2002). Ultrasound causes disruption of
microbial cell walls, membranes, and DNA, by free radical production (Scouten and
Beuchat, 2002; Hulsmans et al., 2010).

15.5  POSTHARVEST QUALITY OF ORGANIC PRODUCE

Irrespective of the health awareness concerns, the continuing increase in the con-
sumption of fresh organic produce can be attributed to the near absence of synthetic
pesticide residues when compared to conventionally grown commodities (Crinnion,
2010; Willer, 2011). In addition, there has been considerable interest in the effect of
organic production methods on bioactive compounds that are being positively associ-
ated with human health benefits. Plant bioactive metabolites are produced by a plant
in response to various stimuli, including pest and environmental stresses (Brandt
and MØlgaard, 2001). These substances are an important source of antioxidant com-
pounds or “antioxidants,” and can be divided into nonnitrogenous compounds such
as phenolic acids, flavonoids, and terpenoids (e.g., carotenoids, xanthophylls), and
nitrogen-containing compounds such as alkaloids, amines, nonprotein amino acids,
glycosides, and glucosinolates (Ölmez and Särkka-Tirkkonen, 2008).
However, despite numerous studies, there is no consensus on the effect of organic
production on levels of bioactives. Table 15.1 summarizes the findings from some
of the studies showing the range of effects on antioxidant levels in a range of pro-
duce. To this list could be added the positive effect of phytonutrients reported by
Worthington (2001), Zhao et al. (2006), Kazimierczak et al. (2008), and Crinnion
(2010), while no significant differences or lower levels were reported by Rosen,
2010; Smith-Spangler et  al. (2012), Asami et  al. (2003), Rossi et al (2008), Kahu
et al. (2010) and Bogs et al. (2012). The different responses of nutritional content in
organic produce is complicated by interaction with cultivars, farm management, soil
quality, weather conditions, and length of time using organic methods (Crinnion,
2010; Aldrich et al., 2011; Camargo et al., 2011).

15.6 CONCLUSIONS
The demand of organic fresh produce continues to grow dramatically (Falguera
et  al., 2012; Schaack et  al., 2013). Organic fruit and vegetables may have higher
Organic Postharvest Technology 341

TABLE 15.1
Comparison of Nutritional Quality in Fruit and Vegetables Produced from
Organic and Conventional Systems
Produce Results Reference
Peach Higher polyphenol in organic Carbonaro et al. (2002)
Apple Higher polyphenol in organic Weibel et al. (2000)
Similar polyphenol content Briviba et al. (2007)
Higher polyphenol in organic Bogs et al. (2012)
Similar antioxidant capacity
Kiwifruit Higher ascorbic acid and total Amodio et al. (2007)
phenolic in organic
Orange Higher ascorbic acid in organic Masamba and Nguyen (2008)
Melon Higher ascorbic acid in organic, but Salandanan et al. (2009)
inconsistency in phenolic content
Blueberry Higher total phenolic, anthocyanin Wang et al. (2008)
and antioxidant activity in organic
Strawberry Higher anthocyanin in organic Camargo et al. (2011)
Persimmon Higher β-carotene in organic Cardoso et al. (2011)
Lower dehydroascorbate
Similar lycopene content
Tomato Lower polyphenol in organic Barrett et al. (2007)
Higher flavonoid and kaempferol Mitchell et al. (2007)
Similar content of nutrient Pieper and Barrett (2008)
Pak choi Higher polyphenol in organic Young et al. (2005)
Lettuce Similar polyphenol content Young et al. (2005)
Similar ascorbic acid Masamba and Nguyen (2008)
Cabbage Similar ascorbic acid Masamba and Nguyen (2008)
Carrot Similar ascorbic acid Masamba and Nguyen (2008)
Broccoli Higher total phenolic content and Aldrich et al. (2011)
antioxidant capacity in organic

levels of antioxidants, although many studies demonstrate no difference between

organic and conventional crops (Dangour et al., 2009; Hoefkens et al., 2009; Smith-
Spangler et al., 2012).
Postharvest handling practices have not changed dramatically with the expansion
of organic fruit and vegetable production over the last decade. Organic packers and
handlers are becoming more aware of the special requires of organic commodities
and greater emphasis on product quality and safety could be anticipated in the future.
There is a need to evaluate fruit and vegetable varieties for better suitability for
organic production systems and postharvest quality.
Fraud is another area of concern as the organic market expands. Documentation
and traceability, especially in the postharvest logistics chain, is especially important,
to avoid mislabeling and a loss of consumer confidence. Though it is a nonbiologi-
cal aspect to the postharvest handling of organic fruits and vegetables, traceability
systems need to be integrated in an effective manner.
342 Advances in Postharvest Fruit and Vegetable Technology

ACKNOWLEDGMENT
We thank Dr. Robert E Paul, University of Hawaii at Manoa, for his helpful sugges-
tions and for reviewing this chapter.

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 16 Modeling in Postharvest
Horticulture
Maarten L.A.T.M. Hertog and Bart M. Nicolaï

CONTENTS
16.1 Introduction................................................................................................... 347
16.1.1 Postharvest and Modeling................................................................. 347
16.1.2 Inductive versus Deductive Models................................................... 349
16.2 Variation in the Data...................................................................................... 350
16.2.1 Treatment Variation........................................................................... 350
16.2.2 Technical Variation............................................................................ 351
16.2.3 Biological Variation........................................................................... 352
16.2.4 Pooling of Variation........................................................................... 352
16.3 Inductive Modeling Approaches.................................................................... 353
16.3.1 Classical Statistics............................................................................. 353
16.3.2 Multivariate Statistics........................................................................ 354
16.4 Deductive Modeling Approaches.................................................................. 355
16.4.1 Modeling Supply Chain Logistics..................................................... 356
16.4.2 Modeling the Postharvest Environment............................................ 356
16.4.3 Modeling Produce Behavior.............................................................. 357
16.4.3.1 Produce Models and Biological Variance........................... 357
16.4.4 Modeling Produce Morphology......................................................... 358
16.4.5 Modeling Produce Metabolism......................................................... 359
16.5 Perspectives of Integrated Models for Postharvest Optimization................. 361
References............................................................................................................... 363

16.1 INTRODUCTION
16.1.1 Postharvest and Modeling
Postharvest research is relatively young and has been rapidly increasing since the
1980s (Figure 16.1). Its main objective is to reduce postharvest losses by enlarging
insight in the underlying physiological process to understand the responsible mecha-
nisms. As highlighted in the previous chapters, thanks to enhanced insights in the
underlying mechanisms, advanced storage conditions, and postharvest treatments
are being developed to continuously improve the postharvest handling chain. Given
the complex nature of the biochemistry and physiology driving the metabolism of
living produce during postharvest, it is not always evident how to interpret the exper-
imental results. Mathematical modeling can help to gain insights in the mechanisms

347
348 Advances in Postharvest Fruit and Vegetable Technology

# Modeling publications in postharvest

# Postharvest publications 1000 100

800 80

600 60

400 40

200 20

0 0
1984
1986
1988
1990
1992
1994
1996
1998
2000
2002
2004
2006
2008
2010
2012
FIGURE 16.1  Number of published items retrieved from Web of Science (All databases)
using the keyword “postharvest” either in or not in combination with the keyword “model” in
the topic field (status on May 01, 2014).

underlying the observed phenomena by screening hypothetical conceptual models

for their fitness to explain the data. The main purpose of modeling in postharvest
is often not the model itself, but the process of modeling, in which the researcher is
forced to line up concepts to bring order in the chaos. A wide range of mathemati-
cal models has found their application in the wider food area (Tijskens et al., 2001)
and the importance in the field of postharvest research can be appreciated from the
increasing contribution of modeling papers (Figure 16.1).
In biological systems, models are, by definition, a simplification of the real world
and, again by definition, will never be true as the only true model is the system itself
(Oreskes et al., 1994). In the current context, a mathematical model is defined as an
abstract representation expressed in terms of mathematical and/or logical constructs.
These can include all sorts of equations (e.g., algebraic, ordinary and partial differ-
ential, and integral equations), different types of Boolean expressions (e.g., proposi-
tions, predicates, and fuzzy logic), or rule-based expressions (e.g., decision tables,
finite state machines and Turing machines) (Nicolai et al., 2005). So far, only few
have been applied to postharvest situations. Once a system has been modeled using
mathematical constructs, relevant real-life actions affecting the system can be substi-
tuted through quantitative reasoning studying their impact. This allows the capture of
relations that are impossible to effectively describe otherwise (Staub and Stern, 1997).
When developing mathematical models to describe the behavior of living pro-
duce, one needs to find a balance between two extremes. One extreme is to describe
all processes in detail. This will inevitably lead to the limitations of current knowl-
edge and will result in a theoretical model that is impossible to parameterize. The
other extreme is to leave out too much detail, simplifying the underlying mecha-
nisms to relationships that only hold for the current situation. In the end, the level of
complexity of the model chosen should match the objectives of the model.
Modeling in Postharvest Horticulture 349

If the objective of the model is to facilitate sorting of fruit by ripeness, a simple

Boolean model could be used, linking fruit color to fruit ripeness, providing a color
threshold above (or below) which the fruit can be considered ripe. If the objective
is to optimize postharvest climacteric ripening conditions in order to deliver ready-
to-eat fruit to the market, a more complex model is needed, quantitatively linking
applied ethylene levels and ripening temperatures to the dynamics of postharvest
fruit-ripening. If the objective would be to develop new varieties with specific ripen-
ing properties, even more detailed mechanistic models might be required, modeling
fruit-ripening control at the metabolic, enzymatic, and/or molecular level. In this
way, a hierarchy of models with increasing complexity can be constructed. In gen-
eral, models with the lowest complexity are often more generic, flexible to transfer to
similar situations, and, therefore, sufficient for management decisions. With increas-
ing level of complexity, models become more specific, can be less readily extrapo-
lated to other situations and conditions without considerable expert knowledge, but
are more useful in generating fundamental insight and understanding of the underly-
ing biology and physics.
In functionalizing the model objective, one will be forced to reduce the available
data to the essentials to be covered by the model. In this context, it is important to
realize that data reduction invariably leads to loss of information. In many cases, the
information filtered out may be not useful (e.g., measurement noise) or not essential
(e.g., aroma data when modeling the softening of mango fruit), but in other cases it
might decrease the accuracy of the model in representing reality (e.g., by ignoring
information on fruit maturity stage when modeling climacteric fruit ripening). To
guide this process of data reduction, proper expert knowledge is essential.

16.1.2 Inductive versus Deductive Models

The different modeling approaches can be roughly separated into two groups, induc-
tive and deductive modeling techniques. With inductive modeling techniques, the
model is induced by the data and no explicit expert knowledge is required. These
techniques are completely data-driven and might result in some generally valid
relationships. Examples of these techniques are the traditional statistical techniques
and modern data mining techniques like neural networks. The deductive modeling
techniques start from the underlying processes, are based on explicit expert knowl-
edge, and are constructed using fundamental laws and generally valid relationships.
Starting from these basic building blocks, the model is constructed and calibrated
against the experimental data. Examples of these techniques are models of heat and
mass transfer, biochemical kinetic models, and models of population dynamics.
Often, one differentiates between fundamental or white-box models, and empiri-
cal or black-box models. Whereas the first class is assumed to be built upon well-
established laws of physics and first principles, the latter class is based on statistical
induction. In reality, many of the so-called mechanistic models are effectively phe-
nomenological models that are based on a simplified assumed mechanism far less
complex than the actual underlying process. For example, to describe respiration
behavior of fruit and vegetables as a function of the oxygen concentration in the
storage atmosphere, often Michaëlis-Menten kinetics, with various modifications to
350 Advances in Postharvest Fruit and Vegetable Technology

account for the effect of temperature and the inhibition by carbon dioxide, have been
developed (Fonseca et  al., 2002; Hagger et  al., 1992; Helena Gomes et  al., 2010;
Hertog et al., 1998; Peppelenbos and van’t Leven, 1996). However, the Michaëlis-
Menten model describes the kinetics of a linear chain of enzyme-mediated reactions
with often a single rate-limiting step while, in reality, the metabolic pathways involved
in respiration are much more complex and cyclic in nature, as can be retrieved from
the kyoto encyclopedia of genes and genomes (KEGG) pathways (Ogata et al., 1999).
Although Michaëlis-Menten kinetics has been shown to describe respiration well, it
must be considered, at most, as a semi-mechanistic or gray-box model.
Metabolic network models take this approach several steps further by providing a
more detailed mechanistic overview by including the detailed stoichiometry of most
important biochemical pathways. While such metabolic models can be detailed in
their biochemistry, they have their own assumptions—for instance, by assuming no
spatial concentration differences of metabolites inside the object of study. While this
is a reasonable assumption at the cellular level, it is not necessarily the case at the
tissue or whole-fruit level. The obvious examples where this homogeneity assump-
tion does not hold, are the case of compartmentalization within plant cells (Tiessen
et al., 2012), the often observed concentration gradients along a tissue differentiation
axis (Macklon and DeKock, 1967), and gas gradients related to the microstructure
of the fruit (Ho et al., 2011).
This example clearly shows that fundamental knowledge on the underlying
mechanisms allow us to move away from purely explorative, statistical approaches
by gradually incorporating more of the available knowledge into the model struc-
ture. Still, in spite of containing more knowledge, detailed mechanistic models
are not always more suitable given computational restrictions, and given the dif-
ficulty of properly calibrating all parts of such complex model. However, it is easy
to foresee that their future scope in ongoing optimization of postharvest systems is
considerable.

16.2  VARIATION IN THE DATA

All experimental data collected in a postharvest context is affected by different
sources of variation (De Ketelaere et al., 2006), some willingly imposed, some inevi-
table, some unknowingly, and some avoidable. When modeling such experimental
data, using either statistical or mechanistic modeling approaches, this variation can
hamper the interpretation of the data, if not properly accounted for.

16.2.1 Treatment Variation
Treatment variation is the variability induced by the independent treatment variables
controlled by the experimenter. When modeling the data, these variables will be
included as part of the model structure to, hopefully, account for the observed dif-
ferences in the postharvest responses. This implies a certain level of experimental
control with the treatment variables being well-defined.
In postharvest literature, one often defines constant set points for the intended
levels of the treatment variables that do not necessarily match the actual fluctuating
Modeling in Postharvest Horticulture 351

values. If the temporal fluctuations are limited, one might be able to assume these
conditions to be constant. However, while 0.5°C difference at room temperature
might be acceptable, the same accuracy during cold storage might make the differ-
ence between either severe or no incidence of chilling injury. In such situations, it is
better to include the actual measured values when modeling the data as compared
to taking the set-point values. Such dynamically changing conditions do, of course,
impose restrictions on the modeling approach that can be applied, but also creates
opportunities, as will be shown later. Similar considerations can be made when spa-
tial variation is involved. When storing packed fruit, conditions in the storage room
atmosphere might not resemble the conditions at the individual fruit level. For this
reason, one should try to take the actual conditions the fruit is exposed to as an input
to analyze the fruit behavior. Alternately, one can extend the model to include the
transport phenomena governing the produce environment.
This concept of replacing assumed constant conditions by either actual measure-
ments or by their modeled counterparts can be extended to the various organiza-
tion levels at which postharvest experimentation takes place (temporal and spatial
variation in, e.g., storage room conditions, dosing levels of postharvest chemicals,
temperature and gas composition inside a fruit, and substrate levels, pH and gas
composition of experimental liquid media). Applying this concept will improve the
quality of the subsequent model analyses, as more of the observed variation can be
attributed to the experimental variables. Any of the treatment variation left unas-
signed will result in unexplained variation in the studied postharvest responses.

16.2.2 Technical Variation
Technical variation is the variability due to measurement error. Some of this error
is random variation inherent to the methodology applied, but often this error can be
related to the design of the experiment. For example, many methodologies depend
on calibration of equipment, stock solutions of reagents, multiple instruments of
the same type (e.g., pipettes, loggers, balances, and climate chambers), or different
brands of consumables that are prone to vary between batches or are sensitive to
drifts due to fluctuations in temperature, humidity, atmospheric pressure, and more.
For this reason, it is important to be aware and track any such relevant variables to
understand changes in the measured responses between, for instance, measurement
days. If this information is available, it can be taken into account during the data
analysis by including such effects as separate factors. If this is practically unfeasible,
the least one should do is to prevent unwanted technical variation from overlapping
with the expected treatment variation by randomizing the measurements of samples.
For instance, when comparing the effect of two temperatures on fruit quality, one
should not analyze samples from one temperature on one day (or using one instru-
ment, or stock solution) while analyzing samples from the other temperature on a
second day (or using another instrument, or stock solution). In such cases, one can
never be sure if the observed effect was due to temperature or technical variation
between the measurement days. In combination with randomization, the inclusion
of reference samples and replication of the regular samples is essential to obtain a
proper measure of the technical variation.
352 Advances in Postharvest Fruit and Vegetable Technology

16.2.3 Biological Variation
Biological variation is the variability arising from the fact that no two biological
items are identical. Most of the time, postharvest management aims at controlling
the average batch behavior and limiting biological variation as much as possible
by sorting and grading the product at the different stages in the postharvest chain.
Biological variation of tomatoes, for instance, becomes discernible through their
initial color at harvest. Assuming all tomatoes go through the same developmental
ripening process from fruit set to fruit senescence, the initial color at harvest can be
interpreted as a measure of the biological age of an individual tomato (Van de Poel
et al., 2012). If all fruit could be harvested at the same maturity, variation at harvest
would be negligible and would remain negligible throughout the postharvest period.
As fruit are not harvested at such a homogenous stage, variation does exist both at
harvest and during postharvest storage (Tijskens and Wilkinson, 1996). Depending
on the underlying mechanism, the biological variation at harvest can remain the same
or can be transformed during postharvest storage. In general, postharvest research
tends to take individual fruit behavior as an unavoidable nuisance and works mainly
on the averaged fruit behavior. With such an approach, biological variation is reduc-
ing the statistical power of the model analysis (Tijskens et al., 2003). Over the years,
alternatives have been developed to integrate biological variation as an explicit part
of the modeling approach enhancing the interpretation of the data (as reviewed by
Hertog et al., 2007a; Jordan and Loeffen, 2013).

16.2.4 Pooling of Variation
One approach often observed in postharvest research to reduce variation in the data
is the pooling of experimental data. This can be done physically by collecting a
single measurement per sample that was pooled by combining multiple sub-samples
(e.g., ethylene production by a pooled group of multiple fruits, enzyme activity in a
tissue sample collected from multiple fruit, or titratable acidity on fruit juice col-
lected from a batch of fruit), or virtually by collecting data as measurements on
the individual fruit and then averaging the values. In both cases, the measurement
represents the average value for a batch with the biological variation completely
removed. When interpreting such pooled data, no information is available to test for
a significant effect of the treatments applied. To do so, one needs to check whether
the effect induced by the treatment is larger than the variation within a treatment
(Cumming et al., 2007). However, this information has been filtered out by the pool-
ing. Even when replicate pooled measurements are collected, these will mainly mir-
ror the technical measurement variation resulting in over-optimistic interpretation
of the data.
The golden rule should be not to pool any data at all, as valuable information
is discarded, thus inhibiting correct scientific interpretation of the data. The only
case in which pooling is inevitable, is when a single fruit would not provide enough
material for the planned analyses. However, in that case, one should at least collect
replicate measurement (on different pooled samples), and be aware of the limitations
of the data when interpreting the results.
Modeling in Postharvest Horticulture 353

16.3  INDUCTIVE MODELING APPROACHES

Inductive modeling techniques are largely data-driven and do not require expert
knowledge to build the models. Often, these statistical techniques are applied just
because expert knowledge is lacking and one mainly tries to understand which fac-
tors might affect the studied response.

16.3.1 Classical Statistics
The most common “model” applied in postharvest is that of a normal distribution.
When comparing treatment effects using the commonly used Student’s t-test, the
underlying assumption is that the data follows such normal distribution. When com-
paring treatments in postharvest research, the number of groups to be compared
quickly increases. Analysis of variance (ANOVA) overcomes this issue by general-
izing the t-test to more than two groups (Payne, 2014). The treatment effects are cal-
culated by partitioning the observed response variance over the treatment factors and
comparing the mean of a treatment to the general mean over all treatments. When
more than one treatment variable is involved, the model underlying the ANOVA can
become more extended with the response variable being interpreted as the combined
effect of the various treatments, including possible interaction terms.
In postharvest studies, the treatment factors might be either well-controlled nonran-
dom factors (so-called fixed effects) or factors still prone to some level of randomness
(so-called random effects). Both situations are included in a special class of ANOVA
models referred to as mixed effects models (Molenberghs and Verbeke, 2000). If the
assumption of normality is violated, generalized mixed models provide the ultimate
alternative, allowing the use of other (user-defined) distributions (Payne, 2014). By
explicitly taking into account random sources at the treatment level, less variation will
remain unexplained when interpreting the dependent postharvest responses.
Besides studying the effect of various postharvest treatments, time plays an
important role in postharvest research as well as in trying to explain the treatment
effect throughout storage and shelf-life. The (generalized) mixed model approaches
can take into account such repeated measurements, typically showing a strong cor-
relation for a single fruit, while being independent between the individual fruit.
Given their additional capability to account for the appropriate sources of biological
variation, (generalized) mixed model approaches are widely applicable in the area of
postharvest operations (Lammertyn et al., 2003a).
A special class of postharvest data that should be treated in a different way is that
concerning the incidence of diseases or disorders, often relying on countable data,
and measured in a binomial manner as either a “yes” or a “no” (often coded as 1 or
0) response. Similar data is obtained when working on consumer acceptance, result-
ing in an “accept” or a “reject” response. Such data can be properly analyzed using
so-called logistic regression (Lammertyn et al., 2000) in which the dependence of
the response on the treatment factors is modeled through probability scores. This
approach can be seen as a special case of generalized models.
Once ANOVA-based techniques have been applied to determine which treatment
factors significantly contribute to the observed differences between groups, the same
354 Advances in Postharvest Fruit and Vegetable Technology

underlying model structure can be used as a regression model to predict the posthar-
vest response as a function of the imposed treatments. By developing such regression
models, the incorporated treatment-factors do have their proven relevance based on
the experimental data. Of course, other regression models can be developed in a
complete empirical way by simple curve fitting and might describe the data even
better, but such empirical models cannot claim any scientific experimental basis.
Reviewing the usage of these various statistical techniques in the area of post-
harvest operations, ANOVA was found to be applied in 35% of all articles published
in the journal named Postharvest Biology and Technology (as on May 01, 2014),
while the t-test was applied in 11% of all published manuscripts. On inspection it
appears that ANOVA is often applied just to obtain the results of the multiple com-
parison t-tests that are commonly available as a posthoc test with ANOVA, without
the actual ANOVA results being presented. The application of (generalized) mixed
model approaches and logistic regression was limited to less than 2% of all publica-
tions in Postharvest Biology and Technology. The application of this last group of
models is clearly hampered by their conceptual complexity as compared to the basic
ANOVA and multiple t-tests. With the increased availability of (generalized) mixed
model tools in most standard statistical software packages, the door is, however, open
for a more intensive application of these advanced statistical modeling techniques.

16.3.2 Multivariate Statistics
Multivariate statistics is often used synonymous with chemometrics, which has its
origin in analytical chemistry. Chemometrics was defined as “How to get chemi-
cally relevant information out of measured chemical data, how to represent and
display this information, and how to get such information into data” (Wold, 1995).
Multivariate statistical methods are extremely suitable to extract information from
large datasets. They can, for instance, be used to profile complex (chemical) produce
properties, to find relationships between produce composition and sensory proper-
ties, to discriminate between cultivars and species, or to detect adulteration in food
products. Especially with the development of data-rich measurement techniques like
the various spectroscopic techniques (e.g., nuclear magnetic resonance, UV, visible,
and near infrared spectroscopy, Fourier transform infrared spectroscopy, Raman
spectroscopy) and the various “omic” techniques generally applied in the framework
of systems biology (Hertog et al., 2011), the application of multivariate statistics has
a wide potential field of application.
Central to most chemometrics is the concept of reducing the complexity of the
data by organizing the original measured variables into groups of highly correlated
variables, with each group being represented by a single new synthetic variable
(referred to as a principal component or latent variable). These principle components
are defined in such a way that they show no mutual correlation, with all redundant
information from the original data being removed, and only the unique information
being conserved. By this reduction in the complexity of the data, patterns can be
more easily recognized. As such, multivariate statistics is less about detecting sta-
tistically significant differences, and more about recognizing patterns by examining
relationships among multiple variables at the same time.
Modeling in Postharvest Horticulture 355

The two most prominent techniques applied in chemometrics are principal com-
ponent analysis (PCA) and partial least square regression (PLS). While PCA is an
unsupervised explorative technique to detect the underlying correlation structures
in the data, PLS provides a supervised approach in which prior knowledge on the
underlying latent structure is provided, and where the PLS regression model will try
to explain this latent structure from the multivariate data-set.
A more recent multivariate modeling technique that was introduced in chemomet-
rics is that of artificial neural networks (Marini et al., 2008). These are mathemati-
cal systems developed in analogy to the human brain, which learns by example. An
artificial neural network can be seen as a black-box that receives multiple inputs and
produces multiple outputs with the in- and out-puts being interconnected through
layers of parallel connected simple arithmetic units (neurons). Parameterization of
the neural network takes place by training using representative examples. Artificial
neural networks are highly suitable for classification purposes.
Chemometrics has been applied in postharvest research, especially in the field of
visual and near infrared spectral data analysis, to link spectral features to the chemi-
cal composition of fruit and vegetables to establish nondestructive measurement
techniques for produce properties like dry matter content, acidity, starch content,
and soluble solids content (as reviewed by Nicolai et al., 2007). The same approach
has been applied with various degrees of success to describe derived quality attri-
butes like firmness (Bobelyn et al., 2010), incidence of internal disorders (Magwaza
et al., 2012), fruit maturity (Peirs et al., 2005), and sensory evaluation (Parpinello
et al., 2013). The application of chemometrics accounts for about 12% of all publica-
tions in Postharvest Biology and Technology.
With “omic” applications still being under-represented in postharvest research
(about 2% of all publications in Postharvest Biology and Technology), chemometrics
for “omics” data still plays only a minor role (Pedreschi et al., 2007; Rudell et al.,
2009; Vandendriessche et al., 2013). Other applications can be found in research in
the area of postharvest volatiles (Ciesa et al., 2013; Vandendriessche et al., 2012),
where the complex aroma composition can be linked to sensory evaluation (Berna
et al., 2005; Obando-Ulloa et al., 2009). Within postharvest research, artificial neu-
ral networks are sporadically used as pattern recognition models to classify produce
according shelf-life, maturity, or the incidence of various defects (ElMasry et  al.,
2009; In et al., 2009; Schouten et al., 1997).

16.4  DEDUCTIVE MODELING APPROACHES

As soon as inductive models have identified some leads to how the studied process
might function, conceptual models will evolve which can be taken as a starting point
for developing deductive models. The basic strategy to develop deductive models
is to apply a systematic process of problem decomposition, dissecting the problem
into its basic building blocks, subsequently reassembling them while leaving out
unnecessary detail. What is essential and what is redundant depends largely on the
intended application of the model. Going through this process of modeling, one is
forced to think about cause and effect. The modeling process helps to structure ideas
and helps to turn them into sound conceptual models. The final mathematical model
356 Advances in Postharvest Fruit and Vegetable Technology

can be used to test these concepts and check their validity, given the current level
of knowledge. The mathematical models can combine expertise from different sci-
entific disciplines and can be targeted to different applications and organizational
levels. Examples can be found in the area of economics, operations management,
logistics, heat and mass transfer, reaction diffusion models, and biochemical path-
way models.

16.4.1 Modeling Supply Chain Logistics

Within postharvest systems, there are many processes taking place at various orga-
nization levels that can benefit from modeling. This starts at the level of supply
chain logistics. In the fresh-food supply chain, one has to consider many factors such
as time-to-market, traceability, transport/storage conditions, handling, production/
process control, demand variability, seasonal behavior, all of which affect the opera-
tion of the supply chain. Fresh-food supply chain model models can be applied to
optimize the chain with regard to speed of operation, cost reduction, and optimized
shelf-life (Dabbene et al., 2008a,b; Hertog et al., 2014; Rong et al., 2011). Such logis-
tic models focus on the timely distribution of the product to satisfy the needs in the
chain and to optimize warehouse management. Common strategies aim at efficient
product management across the distribution chain through FIFO (First-In-First-Out)
or FEFO (First-Expired-First-Out). FIFO is the more commonly adopted approach
while FEFO is more dedicated to perishable products as it will only ship products
depending on the potential of their shelf-life in relation to their end-destination (East,
2011). This requires proper insight in product behavior in response to the conditions
imposed (De Baerdemaeker et al., 2006).

16.4.2 Modeling the Postharvest Environment

While, eventually, the main interest is on the produce itself, its postharvest fate is
largely defined by the correct functioning of the infrastructures involved during har-
vesting, transport, storage, cooling, drying, packaging, and so on. For this reason,
much effort still goes toward optimization and improved design of this posthar-
vest infrastructure through engineering models (Ambaw et al., 2013). The ultimate
aim is to create the best possible environment for the produce under study, which
also requires proper knowledge on produce requirements concerning their storage
conditions.
To model the produce environment, the main emphasis is on the flow, heat, and
mass transfer processes responsible for the temporal and spatial changes in tem-
perature, in the main gas conditions (being the levels of oxygen, carbon dioxide,
and water vapor), and in any additional compounds introduced in the storage atmo-
sphere to control, for instance, postharvest ripening (e.g., ethylene and 1-methylcy-
clopropene), or to suppress tuber-sprouting or the incidence of diseases or fungal
and bacterial growth (Ambaw et al., 2012; Delele et al., 2009; Nahor et al., 2005).
Generally computational fluid dynamics (CFD) approaches are applied as they take
into account both the 3D geometry of storage rooms and produce stacking, and the
numerical calculation of fluid flow and heat and mass transfer, based on fundamental
Modeling in Postharvest Horticulture 357

laws of physics, thus allowing for the incorporation of processes like gas diffusion,
kinetics, and droplet or particle dispersion (Norton and Sun, 2006). While such mod-
eling can be performed starting from the appropriate engineering expertise as such,
most added value is obtained when properly embedded within the multidisciplinary
context involved in postharvest operations.

16.4.3 Modeling Produce Behavior

Most published produce quality models are phenotypic models that describe post-
harvest-produce behavior in terms of responses measured at the whole-fruit or veg-
etable level, such as a change in firmness (Schouten et al., 2010), color (Lana et al.,
2006), sugar content (Hertog et al., 1997), or other relevant product properties and
quality attributes. Some of these changes are part of the physiological process of
fruit-ripening while other processes, such as shriveling (Maguire et al., 2000), are
driven by merely physics. Often such models are based on semimechanistic concepts
formulated through kinetic concepts, but not necessarily one-to-one founded in pro-
duce biochemistry. In addition, they assume the produce to be homogeneous and do
not take into account any spatial variation of produce properties or quality attributes.
For instance, fruit-softening has been modeled many times, using various
approaches with the level of detail often determined by the available experimental
data and the purpose in mind. This ranged from purely empirical curve fitting mod-
els describing kiwifruit softening with the main emphasis on the industrial applica-
tion towards batch segregation (Benge et al., 2000; Jabbar et al., 2014), to relatively
detailed models based on a detailed conceptual approach but calibrated on firmness
data only (Schouten et al., 2010; Tijskens et al., 1999b). Other models were defined
closer to the underlying enzyme actions and calibrated on both firmness data and the
actual enzyme activities measured (Róth et al., 2008; Tijskens et al., 1998), while a
last group of models focused merely on a detailed description of the effect of oxy-
gen and carbon dioxide on the fruit-softening rate to describe the effect of modified
atmosphere conditions (Hertog et al., 2001, 2004).
Overall, this type of models has shown itself to be relatively successful in describ-
ing the dynamics of fruit and vegetable quality attributes, as they are detailed enough
to have some generic value, while they are not hampered by an overly detailed
description that would make them hard to calibrate. When developing these semi-
mechanistic models, one should always try to respect basic fundamental conserva-
tion laws and, at the same time, introduce the required simplifications to deliver
valid models that are scientifically useful. One of the more detailed models of this
kind was a semimechanistic model describing the dynamics of mealiness in apple
(De Smedt et al., 2002). This model described measured textural changes related to
mealiness, starting from the underlying cell-wall physiology and the cellular water
management, incorporating both simplified biochemical reactions as well as the cel-
lular water transfer processes.

16.4.3.1  Produce Models and Biological Variance

What differentiates the postharvest chain from most other handling chains is that
the postharvest chain involves dealing with living tissues that inherently change with
358 Advances in Postharvest Fruit and Vegetable Technology

time and which show large sources of biological variance. For the sake of simplicity,
one generally focuses on understanding the average batch behavior, but from a mar-
keting point of view, one has to deal with the biological variance present. Recently,
new impulses have been given to include biological variation as part of postharvest
product models (as reviewed by Hertog et al., 2007a). One of the driving forces for
this has been the increased availability of nondestructive measuring techniques that
allow monitoring of individual objects during time (De Baerdemaeker et al., 2006;
Nicolai et al., 2014; Tijskens et al., 1999a; Zerbini et al., 2006). By introducing bio-
logical variance into the product models, both the analysis and the subsequent pre-
diction of postharvest batch behavior can be considerably improved as it allows for
predicting the propagation of the initial biological variance at harvest throughout the
whole postharvest chain taking into account all relevant aspects affecting posthar-
vest fruit behavior (Hertog et al., 2008; Schouten et al., 2004). In recent years, more
and more applications have been found where models incorporate biological varia-
tion to allow for optimization of the postharvest handling chain based on the whole
batch behavior (Eccher Zerbini et al., 2009; Guillard et al., 2012; Gwanpua et al.,
2013; Hertog et al., 2007b; Jabbar et al., 2014; Jordan and Loeffen, 2013).

16.4.4 Modeling Produce Morphology

When progressing beyond phenotypic models to create more true-to-life models, a
first step is to include the overall morphology of the produce under study through
its 3D geometry. Such macroscale models have been proven successful to model
the combination of respiration, gas diffusion, and gas permeation (Ho et al., 2008;
Lammertyn et al., 2003b), either in or not in combination with heat transfer (Benítez
et al., 2012). These models allow accounting for spatial differences arising from dif-
fusion processes under the assumption of a homogenous internal structure. However,
this ignores the microscale structure of plant tissue arising from tissue differentia-
tion, which contributes greatly to a heterogeneity in heat and mass transfer prop-
erties of the tissue, as the various structures have different characteristics, such
as porosity, connectivity, and conductance (Herremans et  al., 2013; Kuroki et  al.,
2004). Microstructural models have been developed, starting from various imaging
techniques to provide realistic tissue geometries, either using simple 2D reconstruc-
tions (Ho et al., 2009), or based on more detailed 3D reconstructions (Herremans
et al., 2013). A 3D microscale model of a complete fruit is, however, not yet feasible
because of its excessive computational requirements. For this purpose the multiscale
modeling paradigm is applied, which combines continuum-type macroscale models
with microscale description of features in the regions of interest, in this way creating
a hierarchy of models (Ambaw et al., 2013; Ho et al., 2013; Figure 16.2). The models
are coupled via multiscale analysis, in which the model results relevant to the mac-
roscale are linked to simulations at the microscale by means of homogenization and
localization procedures. Such a multiscale approach has been extensively applied
to model gas-exchange of fruit (Ho et al., 2011) and to model mechanical proper-
ties of whole fruit and plant tissues (Ghysels et  al., 2010; Li et  al., 2013). Instead
of using real-imaged geometries, there is also the possibility to start from gener-
ated virtual geometries mimicking real tissue (Abera et al., 2013; Fanta et al., 2014;
Modeling in Postharvest Horticulture 359

FIGURE 16.2  Artist’s impression of the multiscale modeling approach applied to apple. At
the microscale model a computational tomographic image of apple fruit tissue was used to
generate a 3D geometry of the cortical fruit tissue. Based on simulations at this microscale
tissue properties of gas permeability were estimated. These diffusion properties were applied
to a homogenized lumped model at the whole-fruit scale under the assumption that all parts
of the cortex have the same diffusion properties. Based on this assumption, gas gradients at
the whole fruit level were calculated.

Mebatsion et al., 2009; Pieczywek et al., 2011). The added advantage of including

realistic geometries obviously lies in accounting for detailed spatial differences
induced by the geometry affecting, through the governing physics of heat and mass
transfer, the functioning of the plant’s metabolism.

16.4.5 Modeling Produce Metabolism

To quantitatively understand the functioning of fruit metabolism, the phenotypic
models have to be extended towards the relevant metabolic pathways involved and
their control. This control can be at the metabolite level (e.g., substrate availability
and product inhibition), at the enzymatic level (e.g., reaction kinetics, allosteric regu-
lation, enzyme turnover, and posttranslational modification), and at the transcription
level (e.g., enzyme induction, switching between specific isoforms and gene silenc-
ing). While the main metabolic pathways are often well-defined, the various control
systems are less well-known, limiting the development of system-wide regulatory
models (de Jong, 2002).
The most essential part of plant metabolism governing postharvest maintenance
of fruits and vegetables is respiration (e.g., glycolysis, Krebs cycle, and pentose
phosphate pathway). Respiration releases the energy enclosed in carbon compounds
for maintenance, and generates carbon precursors for biosynthesis. While at the plant
or plant organ level, respiration has been mainly modeled through strongly simplified
Michaelis-Menten models (e.g., Hertog et al., 1998), detailed fundamental modeling
approaches have been applied to unicellular organisms such as yeast (Herrgård et al.,
2008; Teusink et al., 2000). More recently, these efforts have been extended to simple
plant systems such as Arabidopsis thalliana (Szecowka et al., 2013; Williams et al.,
360 Advances in Postharvest Fruit and Vegetable Technology

2010). The difficulty with the application to multicellular plant systems is that: (1)
inside plant cells, metabolic reactions are spatially distributed over multiple cellular
compartments; and (2) tissue differentiation has resulted in multiple cell types, each
having their own metabolic characteristics. These two aspects require the introduc-
tion of transport terms between cells and between cellular compartments.
To make such extensive metabolic models work, kinetic information is required
on the involved enzymes and transporters. Most literature data on enzyme activity
is based on in vitro measurements under ideal conditions, concerning factors such as
substrate availability, pH, temperature, and cofactors. Such theoretical enzyme activ-
ities no longer hold in vivo and have to be estimated from the experimental data on in
vivo fluxes. When dynamic responses are studied, the time profiles of the observed
changes are largely dictated by the underlying enzyme specific kinetic mechanisms.
As these mechanisms are often not known, steady state approaches are taken. When
a system is at steady state, the actual kinetic mechanism is no longer relevant and
can be omitted from the model. While these simplified steady state models are easier
to derive, their application is inherently limited to steady state situations only; no
conclusions can be drawn on, for instance, how fast a fruit will adapt to an applied
postharvest condition, only on the eventual steady state itself. Given the dynamic
nature of fruit ripening and senescence, the steady state assumption can only be
valid when relative short time periods are considered. When evaluating dynamic
postharvest conditions, steady state cannot be assumed and the full dynamic changes
have to be included. Where the underlying kinetics are not known, they will have to
be estimated from experiments designed to excite the system in such a way that it
reveals its characteristic dynamic behavior. While the first steps have been taken to
develop detailed models on the main respiration pathways, some effort is still needed
to extend these models to the level of intact fruits and vegetables.
Of special postharvest interest is the plant hormone ethylene, which is involved in
many physiological processes, including the ripening of climacteric fruit. Over the
years, various models describing ethylene biosynthesis have been developed, going
from schematic representations, including the importance of different enzymatic
isoforms (Alexander and Grierson, 2002), to elaborated Michaelis-Menten kinetics
to describe the activity of ACO (Sanders and de Wild, 2003). A more mechanistic
approach was followed by Génard and Gouble (2005), who developed a theory of
ethylene emission by peach, based on a mathematical representation of the respira-
tion process and ethylene pathway. They incorporated the effect of ambient fluctuat-
ing temperatures through their effect on respiration. A similar model was applied
to apple by East (2007). In all cases, the models were only calibrated and validated
based on experimental data concerning respiration and ethylene production rates,
while all intermediate metabolites and related enzyme activities remained hypotheti-
cal. More recently, a detailed kinetic model of the ethylene biosynthesis pathway was
developed, based on actual quantitative data of all the intermediates and enzymes
involved in the ethylene biosynthesis in CA-stored apple (Bulens et al., 2012), and
ripening tomato fruit (Van de Poel et al., 2014) starting from the level of transcrip-
tomics (Figure 16.3). Over the years, models of the ethylene biosynthesis have pro-
gressively included more detail, integrating aspects from the different organization
levels but without considering the spatial organization within the cell or tissue. What
Modeling in Postharvest Horticulture 361

Cell wall/membrane
kdiff

MACC
RNAMTN MACCT
Ethylene
kt,MTN kpd,MTN
DACC kMACC
MTNp kDACC
MTA kACO ACO
kMTN ACC kt,ACO
kpd,ACO
Polyamines kACS2,4,6
dSAM SAMdc RNAACO1
kSAMDC ACS
Yang cycle

SAM

kpd,ACS2,4,6

Genes ACS2 ACS4 ACS6

Literature
input
Proteins kt,ACS kt,ACS kt,ACS
Data
Metabolites input RNAACS2 RNAACS4 RNAACS6

FIGURE 16.3  Visual representation of the mathematical model developed by Van de Poel
et  al. (2014) describing the ethylene biosynthesis pathway for ripening tomato fruit. Solid
arrows represent direct conversions, while the dotted arrows indicate more general pathway
interactions containing multiple conversion steps. Ethylene is the end-product of a cascade of
enzymatic reactions and diffuses out of the cellular tissue through the cell wall/membrane.
The enzyme levels result from both gene expression and protein degradation.

remains is the inclusion of the binding kinetics of ethylene molecules to the ethylene
receptors and the downstream signaling cascade leading to the activation of multiple
ethylene response factors and the associated physiological responses such as fruit
softening (Bennett and Labavitch, 2008).

16.5 PERSPECTIVES OF INTEGRATED MODELS

FOR POSTHARVEST OPTIMIZATION
Overall, postharvest modeling allows postharvest systems to be analyzed, predicted
and improved, either in a manual way or through an integrated approach of model
predictive control, in which postharvest management decisions are made based on
model-based prediction of the product behavior (Lukasse and Polderdijk, 2003;
Verdijck et al., 1999). The aim of modeling in postharvest is therefore not to develop
true models but to develop valid models that are consistent with our current knowl-
edge level and that contain no known or detectable flaws of logic. Also, models
should be sufficiently detailed for the intended purpose but, at the same time, simpli-
fied to give robust manageable models.
While the previous sections have summarized an already large volume of model-
ing research, there is still a need and a potential for integrated models to progress
362 Advances in Postharvest Fruit and Vegetable Technology

scientific concepts and support postharvest optimization. These applications can be

found in the area of optimizing the design of packages, shipping containers, and
storage rooms, to create the best possible conditions for postharvest storage, either in
or not in combination with product models, to understand the effect of the governing
conditions on produce metabolism and, eventually, its quality.
Still, the link between what happens at the level of metabolic pathways and what
becomes measurable at the intact fruit level, might not always be fully known. For
instance, fruit firmness is the resultant of complex cell-wall biochemistry (Johnston
et al., 2002). While, over the years, the pathways and the enzyme involvements have
been increasingly unraveled, the challenge remains to translate the biochemical
changes observed at the level of cell-wall polymers (e.g., degree of methylation and
esterification; Brummell, 2006) to the level of the mechanical properties of fruit
firmness. This is complicated by the observation that clear differences exist in the
genetic regulation of fruit-softening between fruits and even between cultivars of the
same fruit (Goulao and Oliveira, 2008). While complicating the transfer of experi-
mental findings, phenomena such as fruit-softening provide a good playing ground
for developing an integrative generic model that, depending on the fruit, is acti-
vated in a different configuration and that, through such variations, can orchestrate
the fruit-specific process of cell-wall breakdown, leading to the softening patterns
characteristic for the selected fruit. In this way, modeling can be used to fine-tune
and develop our understanding of the whole process through the iterative process of
model-based hypothesis development, and the subsequent challenge to these hypoth-
eses through experimental data.
Another important potential application field for modeling in postharvest opera-
tions is that of predictive microbiology, as postharvest shelf-life is often limited by
microbial growth. In addition, fresh fruit and vegetables are nowadays recognized
as providing an important route of entry for zoonotic human pathogens into the
food chain (Brandl, 2006). While a large volume of modeling research exists on
predictive microbiology (McMeekin et al., 2013), it mainly applies to well-controlled
growing conditions, either on artificial growth media or in the framework of fer-
mentation processes, or in relation to the microbial safety of fish and meat products.
Some predictive microbiology has been performed in relation to modified atmo-
sphere packaging of fruits and vegetables either with or not with real produce, and
under actual logistic-chain conditions (Geysen et  al., 2005; Jacxsens et  al., 2002;
Koseki and Isobe, 2005). The extension of predictive microbiology to postharvest
situations is still limited due to the many unknown factors in terms of initial infec-
tion levels, the actual microorganisms involved, and the complexity of fruits and
vegetables as a bio-based growth medium. With the booming of the fresh-cut pro-
duce industry (Nicola et al., 2006), there is an urgent need for quantitative insights
in risk assessments throughout the food supply chain. Ultimately, this requires the
combination of logistic risk assessment models (Tromp et al., 2010) with numerical
tools to include the appropriate sources of variation (Busschaert et al., 2011; Ferrer
et al., 2008; Nicolai and Van Impe, 1996; Poschet, 2003).
In summary, modeling in postharvest operations has two important benefits that
still can be capitalized more profoundly. The first is from a scientific point of view,
in testing different conceptual models on their fitness to explain the data, enhancing
Modeling in Postharvest Horticulture 363

the understanding of what mechanisms could be responsible for the observed phe-
nomena. The second benefit is from an applied point of view, where mathematical
modeling can be used as an instrument to disseminate scientific knowledge to the
wider industry in such a way that it can be readily applied in the form of management
support systems.

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Horticultural / Agricultural Science

Advances in Postharvest Fruit and Vegetable Technology

The postharvest handling of fresh fruits and vegetables plays a critical
role in facilitating a continuous supply of high-quality fresh produce to
the consumer. Many new technologies developed and refined in recent
years continue to make possible an ever-expanding supply of fresh
products. This volume examines a range of recently developed
technologies and systems that will help the horticulture industry to
become more environmentally sustainable and economically competitive,
and to minimize postharvest quality loss and generate products that
are appealing and acceptable to consumers.

Advances in Postharvest Fruit and Vegetable Technology examines

how changes in community attitudes and associated pressures on
industry are demanding changes in the way technology is used to
minimize postharvest loss and maintain product quality. In particular,
the book discusses important drivers for change, including

• Using more natural chemicals or physical treatments to replace

synthetic chemicals
• Increasing the efficiency of older, more traditional methods in
combination with newer biocontrol treatments
• Leveraging a range of biomolecular research tools or “omics” to
efficiently gather and assess mass information at molecular,
enzymic, and genetic levels
• Using modeling systems to identify key changes and control
points for better targeting of new treatments and solutions to
postharvest problems

K22083
ISBN: 978-1-4822-1696-7
90000

9 781482 216967