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POSTHARVEST PHYSIOLOGY OF FRESH-CUT TOMATO SLICES

A thesis submitted to The University of Queensland, Australia in fulfillment of the requirements for the degree of

Doctor of Philosophy in Horticultural Science

By

Darwin H. Pangaribuan

Ir., Bogor Agricultural University, Indonesia PG Dipl., International Institute for Geo-Information Science

and Earth Observation, Enschede, The Netherlands M.Sc., Wageningen University, The Netherlands

School of Agronomy and Horticulture

2005

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DECLARATION OF ORIGINALITY

The work presented in this thesis is to the best of the author’s

knowledge and belief, original and the author’s own work, except as acknowledged in the text and that the material

has not been submitted, either in whole or in part, for a degree at this or any other university.

Darwin Pangaribuan

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LIST OF PRESENTATIONS

5th Australian Horticultural Society Meeting, September 2002, University of Sydney, Australia.

1. Effect of Temperature and Maturity Stage (Poster)

2. The Physiology of Fresh-cut Tomato Slices (Poster) 21st ASEAN/3rd APEC Seminar on Postharvest Technology, August 2003, Bali, Indonesia.

1. The Effect of Fruit Maturity and Storage Temperature on

Postharvest Physiology of Fresh-Cut Tomato Slices (Oral)

2. Biochemical Changes in Tomato Slices as Affected by Fruit

Maturity and Storage Temperature (Poster)

3. Effect of Slicing on Ethylene Production and Respiration Rate of

Tomato Slices (Poster)

Australasian Postharvest Horticulture Conference, October 2003, Brisbane, Australia.

1. 1-Methylcyclopropene Delays Softening in Tomato Slices (Oral)

2. Effect of an Ethylene Absorbent on Quality of Tomato Slices

(Poster)

3. Exposure of Tomato Fruit to 1-MCP Improves Quality of Stored

Slices (Poster)

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List of Publications

Available at:

http://unila.academia.edu/DarwinPangaribuan

https://www.researchgate.net/profile/Darwin_Pangaribuan

No Journal Publications 1 Pangaribuan, D.H., and D. Irving 2011. Effect of maturity and 1 MCP on

quality of tomato slices. Jurnal Agronomi Indonesia Vol. 39(2):92-96

2 Pangaribuan, D.H, D. Irving and Tim O’Hare. 2010. Effect of Maturity and 1-MCP on Physiology of Tomato Slices. Acta Horticulturae No 880:361-366

3 Pangaribuan, D.H and D. Irving. 2010. The Effect of 1-MCP in Maintaining the Quality of Tomato Slices. Jurnal Teknologi dan Industri Pangan, Vol XXI (1): 80-86

4 Pangaribuan, D.H. 2009. The Effect of Ethylene in Maintaining Quality of Tomato Slices. Jurnal Teknologi dan Industri Pangan, Vol XX(1) : 50-55

5 Pangaribuan, D.H. and D. Irving. 2008. Effect of Fruit Maturity and Storage Temperature on the Quality and Storage Life of Tomato Slices. Jurnal Agrista. Vol 12 (1):51 – 61

6 Pangaribuan, D.H. and D. Irving. 2006. The Physiology and Nutrition of Tomato Slices as Affected by Fruit Maturity and Storage Temperature. Jurnal Agrista, Vol 10 (3):142 – 151.

7 Pangaribuan, D.H. and D. Irving. 2006. The Effect of Heat Treatments on the Postharvest Quality of Tomato Slices. Jurnal Agrotropika, Vol 11 (2): 74 – 82.

8 Pangaribuan, D.H. 2006. Ethylene Production and respiration rate in fruit and sliced tomatoes. Jurnal Agrotropika, Vol 11 (1): 15 - 22.

9 Pangaribuan, D.H. 2005. The Physiological Characters of Tomato from Different Portions of Fruit. Jurnal Agrotropika, Vol 10 (2): 101 – 106.

10 Pangaribuan, D.H., D. Irving. 2004. The Involvement of Ethylene in Softening of Tomato Slices. Thai Journal of Agricultural Sciences, Vol.37

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Proceeding Publications

11 Pangaribuan, D. 2011. Effect of Storage Temperature and Ethylene Absorbent on the Quality of Cherry Tomatoes. Proceeding on Seminar Nasional Sains dan Teknologi IV. Lembaga Penelitian Universitas Lampung. Pages 247-256.

12 Pangaribuan, D. 2010. The effect of chemical treatment on the quality of tomato slices. Proceeding on International Seminar on Horticulture for Food Security. ISHFS. Juni 2010.

13 Irving, D. E. and Pangaribuan, D. 2009. The Effects of Cultivar and Storage Temperature on Postharvest Characteristics on Tomato Fruits. Proceeding International Seminar on Sustainable Biomass Production and Utlization: Challenges and Opportunities August 3-4, 2009

14 Pangaribuan, D; D.E. Irving; T,J, O’Hare. 2003. 1-MCP delays ripening in tomato slices. Proceeding of Australasian Postharvest Horticulture Conference, pp. 169 – 171.

15 Pangaribuan, D; D.E. Irving; T,J, O’Hare. 2003. Effect of ethylene absorbent on quality of tomato slices. Proceeding of Australasian Postharvest Horticulture Conference, pp. 252 – 253.

16 Pangaribuan, D; D.E. Irving; T,J, O’Hare. 2003. Exposure of tomato fruit to 1-MCP improves quality of stored slices. Proceeding of Australasian Postharvest Horticulture Conference, pp. 254 – 255.

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ACKNOWLEDGMENTS

I sincerely thank my principal advisor, Dr Donald Irving, for his

valuable guidance, his understanding of an international student to

whom English is a secondary language, and for valuable criticism and

editing during writing each chapter of the thesis. His kindness has

contributed significantly to my learning in the preparation of posters

and seminars, and in the completion of this thesis.

I wish to thank to my co-advisors, Dr Gavin Porter, for his support at

the early stage of the study and Dr Tim O’Hare (Queensland

Department of Primary Industries and Fisheries) and Assoc Prof. Alan

Wearing, for their advice during my study. My gratitude is expressed

especially to Victor Robertson, for help in running laboratory

equipment; Katherine Raymont for help in running the HPLC and

spectrophotometer; Dr Andrew Macnish who helped me with the GC

and in the preparation and quantification of 1-MCP; Alan Lisle for

statistical advice; Leigh Baker (Queensland Department of Primary

Industries and Fisheries) for help running the autotitrater; Darren

Zielke (Syngenta Seeds Pty. Ltd) for helping me to obtain tomato

materials; Julie Hilton (Student Support Advisor) who proof-read part

of my thesis.

Pursuing Ph.D studies in Australia would not have been possible

without a scholarship from the Australian Agency for International

Development (AusAID). Thanks also to the Rector of the University of

Lampung, Indonesia for his permission to pursue postgraduate study.

The completion of this thesis is also supported by my family,

especially my loving wife Dame Trully Gultom, who worked hard to

provide financial assistance, and my son, Daniel Gramy Pratama.

Darwin Pangaribuan bungdarwin@unila.ac.id

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ABSTRACT

Fresh-cut products are becoming increasingly popular as an option for

processing fruit and vegetable commodities. Rapid deterioration

during storage of tomato slices is the main problem with fresh-cut

tomato. Slicing disrupts the plant tissue so the products become

more perishable, which leads to a relatively short storage life, tissue

softening, and results in tomato slices with poor quality. The

scientific basis for maintaining quality of tomato slices during storage,

and postharvest handling techniques to extend storage life, is the

focus of this thesis.

The major research objectives of this study focused on the

physiological (ethylene and respiration), quality (firmness, colour,

soluble solids, titratable acidity, and electrolyte leakage) and

nutritional (ascorbic acid and lycopene) changes that occur in fresh-

cut tomato slices from cv. ‘Revolution’ during storage. The specific

objectives of the research were:

1. To determine the effects of slicing on the postharvest physiology

of tomato slices

2. To study the quality changes in tomato slices taken from fruit at

different stages of maturity and stored at different storage

temperatures

3. To characterise the involvement of ethylene in the loss of slice

quality

4. To determine the efficacy of 1-MCP in maintaining quality of

tomato slices

5. To determine the effect of fruit maturity and 1-MCP on the

quality of tomato slices

6. To evaluate the effect of applying a brief heat shock to intact

tomatoes on the quality of slices.

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Study of the physiology of fresh-cut of tomato slices was started with

comparisons of ethylene production and respiration between intact

tomatoes and sliced tomatoes (arranged stacked or scattered in

storage containers). Ethylene production and respiration initially

increased in response to slicing. The rate of ethylene production and

respiration by tomato slices was higher than in intact fruit. Slices

arranged in stacks had lower rates of ethylene production and

respiration compared with slices that were scattered. To reduce

ethylene production and respiration rates by tomato slices, regrouping

slices into their original shape is desirable during storage.

Tomato fruits at different stages of maturity have different

physiological and metabolic activities when stored at different

temperature regimes. Slices taken from fruit at four stages of

maturity, characterized by colour as ‘turning’, ‘pink’, ‘light-red’, and

‘red’, were evaluated for quality when stored at 0, 5 or 10 °C. The

slices taken from the ‘turning’ stage of maturity were firmer and had

longer storage life compared with those slices taken from the ‘red’

maturity tomatoes. Tomato slices stored at 0 °C were firmer and had

longer storage life compared with those slices stored at 10 °C. Storage

life of tomato slices could be maintained for 12 days at 0 °C, 10 days

at 5 °C, or 8 days at 10 °C. Tomato slices obtained from the ‘pink’ and

‘light-red’ stages of maturity would be acceptable for marketing.

Experiments were conducted to investigate whether ethylene

absorbents and ethylene influence the quality of tomato slices.

Ethylene absorbent resulted in reduced ethylene, less CO2

accumulation, and firmer slices. In contrast, ethylene applied 2 days

after slicing stimulated the rate of ethylene production, CO2

production, and produced softer slices during storage. These

experiments show that endogenous ethylene produced by slicing of

intact tomatoes or application of exogenous ethylene to slices in

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containers had the undesirable effects of inducing softening during

storage.

Changes in firmness are ethylene-mediated in tomatoes and can be

prevented by exposure of fruit to 1-methylcyclopropene (1-MCP).

When intact tomatoes at the ‘pink’ maturity stage were treated with

0.1, 1.0 or 10.0 µL L-1 1-MCP (20 °C, 12 h), 1-MCP reduced both

ethylene production and respiration rate, delayed softening of the

pericarp, and inhibited loss in titratable acidity in slices when

compared with slices from fruit not treated with 1-MCP. The storage

life of tomato slices was extended by application of 1-MCP to intact

tomatoes, but application 1-MCP to slices was of no benefit. The most

effective concentration of 1-MCP for inhibiting the ethylene-induced

softening of tomato slices was 1 µL L-1.

A study was carried out to determine whether 1-MCP would be more

effective if applied at an early maturity stage or a late maturity stage.

1-MCP (1 µL L–1 at 20 °C) was applied directly to intact tomatoes at

‘turning’ and ‘pink’ (early maturity) and ‘light-red’ (late maturity)

stages. The efficacy of 1-MCP was affected by fruit maturity, as later

maturity fruit were usually less responsive to 1-MCP. This study has

shown that application of 1-MCP to intact tomato retarded the

progress of ripening and reduced the rate of loss in slice quality if

applied at the early stages of maturity (‘turning’ and ‘pink’ stages).

The effect of heat treatment on tomato slice quality was determined

when intact ‘pink’ maturity stage tomato fruit were dipped in water at

38 °C, 42 °C or 46 °C for 1 hour or treated with hot air at 38 °C for 24

h, 36 h, or 48 h. Dipping intact tomatoes in hot water or treating

intact tomatoes with hot air prior to slicing did not extend the storage

life of tomato slices.

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This thesis showed that pre-slicing treatments such as selection of

slice portion and arrangement, appropriate fruit maturity and storage

temperature, and application of the ethylene inhibitors 1-MCP, as well

as post-slicing treatments such as ethylene reduction strategies, can

minimise the negative effects of wounding. The information obtained

from this study will provide valuable information for the development

of tomato slice production and marketing.

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TABLE OF CONTENT

1. INTRODUCTION .................................................................... 13

1.1 FRESH-CUT PRODUCTS ................................................................ 13 1.2 PROBLEMS WITH FRESH-CUT PRODUCTS ......................................... 15

2. LITERATURE REVIEW ........................................................... 17

2.1 TOMATO BIOLOGY ...................................................................... 17 2.1.1 Introduction............................................................................ 17 2.1.2 Fruit structure ........................................................................ 17 2.1.3 Fruit composition ................................................................... 19

2.2 FRUIT QUALITY .......................................................................... 20 2.2.1 Introduction............................................................................ 20 2.2.2 Colour .................................................................................... 21 2.2.3 Texture ................................................................................... 23 2.2.4 Sugars ................................................................................... 24 2.2.5 Titratable acidity ................................................................... 25 2.2.6 Flavour ................................................................................... 26 2.2.7 Nutritional value .................................................................... 26 2.2.8 Summary ............................................................................... 28

2.3 FACTORS AFFECTING TOMATO QUALITY ........................................... 28 2.3.1 Introduction............................................................................ 28 2.3.2 Cultural practices .................................................................. 29 2.3.3 Cultivars ................................................................................ 29 2.3.4 Maturity ................................................................................. 31 2.3.5 Low temperature .................................................................... 33 2.3.6 Relative humidity ................................................................... 34 2.3.7 Chilling injury ........................................................................ 35 2.3.8 Summary ............................................................................... 38

2.4 PHYSIOLOGICAL CHANGES DURING FRUIT RIPENING ........................... 38 2.4.1 Introduction............................................................................ 38 2.4.2 Ethylene biosynthesis and production .................................. 39 2.4.3 Respiration ............................................................................ 44 2.4.4 Loss of chlorophyll and synthesis of lycopene ...................... 45 2.4.5 Fruit softening........................................................................ 46 2.4.6 Degradation of starch, and sugar changes ........................... 47 2.4.7 Changes in cellular membranes ............................................ 48 2.4.8 Summary ............................................................................... 49

2.5 CONTROL OF RIPENING ................................................................ 50 2.5.1 Introduction............................................................................ 50 2.5.2 Effect of ethylene on quality attributes .................................. 50 2.5.3 Control of ethylene action ...................................................... 51 2.5.4 1-methylcyclopropene ............................................................ 53 2.5.5 Heat treatment ....................................................................... 56 2.5.6 Summary ............................................................................... 60

2.6 PHYSIOLOGICAL CHANGES AFTER SLICING ....................................... 60 2.6.1 Introduction............................................................................ 60 2.6.2 Wound-ethylene synthesis .................................................... 61

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2.6.3 Elevated respiration............................................................... 63 2.6.4 Membrane deterioration......................................................... 65 2.6.5 Wound healing ....................................................................... 66 2.6.6 Water loss .............................................................................. 66 2.6.7 Susceptibility to micro-organisms .......................................... 67 2.6.8 Loss of firmness ..................................................................... 68 2.6.9 Flavour changes .................................................................... 69 2.6.10 Nutritional changes.............................................................. 69 2.6.11 Summary ............................................................................. 71

2.7 GENERAL SUMMARY ................................................................... 72 2.7.1 Summary ............................................................................... 72 2.7.2 Objectives .............................................................................. 73

3. MATERIALS AND METHODS.................................................. 74

3.1 OVERVIEW ................................................................................ 74 3.2 TOMATO FRUIT .......................................................................... 75 3.3 ASSESSMENTS MADE BEFORE EXPERIMENTATION ............................. 75 3.4 HANDLING OF SLICES .................................................................. 76 3.5 EXPERIMENTAL MEASUREMENTS ................................................... 77

3.5.1 Ethylene and CO2 evolution ................................................... 77 3.5.2 Slice firmness ........................................................................ 79 3.5.3 Soluble solids (SS), titratable acidity (TA) and ratio SS/TA ... 79 3.5.4 Juice colour ............................................................................ 80 3.5.5 Electrolyte leakage ................................................................ 81 3.5.6 Ascorbic acid.......................................................................... 81 3.5.7 Lycopene ................................................................................ 83

3.6 STATISTICS AND DATA ANALYSES ................................................... 84

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1. INTRODUCTION

1.1 Fresh-cut products

Fresh-cut products have been defined as those products arising from

procedures such as washing, sorting, trimming, peeling, slicing, or

chopping, that do not affect the ‘fresh-like’ quality of the fruit or

vegetable (Burns, 1995; Cantwell and Suslow, 2002). A more

encompassing definition for fresh-cut fruit or vegetables was proposed

by Salunkhe et al. (1991b), as “a fresh fruit or vegetable that has gone

through a process to increase its functionality without greatly

changing its fresh-like properties”. Reyes (1996) and Ahvenainen

(2000) defined the requirements of fresh-cut as (1) delivering to the

consumer a fresh-like product and at the same time ensuring food

safety and maintaining sound nutritional and sensory quality, and (2)

ensuring that the product should have sufficient shelf-life to make

distribution feasible within the region of consumption.

Fresh-cut products were previously termed ‘minimally processed’, and

included handling, preparation, packaging and distribution of

agricultural commodities in a fresh-like state (Shewfelt, 1987). The

term evolved into ‘lightly processed’ products (Abe and Watada, 1991;

Huxsoll and Bolin, 1989), to the currently accepted term of ‘fresh-cut’

(Watada et al., 1996). In this study the term ‘fresh-cut’ will be used

throughout, as used in papers on kiwifruit (Agar et al., 1999), pear

(Gorny et al., 2000), and tomato (Hong and Gross, 2000; Hong et al.,

2000).

Production of fresh-cut vegetables and fruits has increased

dramatically in the last decade both as a result of changes in

lifestyles, and in response to consumer demand for convenient, ready-

to-use and ready-to-eat products with fresh-like quality (Ahvenainen,

1996; Garrett, 2002). The International Fresh-Cut Produce

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Association (IFPA) estimated the market value of fresh-cut produce in

the USA to reach approximately $10-12 billion in sales in 2004 (IFPA,

2004).

Fresh-cut products are value-added products that provide an

additional outlet to consumers and could increase the consumption of

fresh produce due to its convenience and attractive appearance and

flavour. For the food service industry, fresh-cut products are

convenient because labour for preparation and special systems to

handle waste is not required. In addition, specific forms of fresh-cuts

can be delivered to the consumer at short notice and there is no

additional preparation for their use in restaurants, fast food outlets

and retail markets (Cantwell and Suslow, 2002; Watada et al., 1996).

Maintenance of quality is a challenge to the rapidly expanding fresh-

cut sector. Maintaining quality of fresh-cut vegetables products has

been possible in carrot (Barry-Ryan and O'Beirne, 1998; Bolin and

Huxsoll, 1991), lettuce (Varoquaux et al., 1996), mushrooms (Brennan

et al., 2000) and pak-choi (O'Hare et al., 1995). Research on apples

(Buta et al., 1999), kiwifruit (Varoquaux et al., 1990), and oranges

(Pretel et al., 1998) has also been reported.

Tomato is a vegetable fruit and little attention has been paid to fresh-

cut tomato slices. Tomatoes have great potential for fresh-cut

processing (Anonymous, 2002a). As tomato slices are a fresh

commodity, there is a need for ready-to-eat tomato slices that have an

acceptable condition for the food service industries, for use on

sandwiches, burgers and in restaurants.

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1.2 Problems with fresh-cut products

Fresh-cut products remain biologically and physiologically active, as

the tissues are living and respiring (Reyes, 1996). The main problems

with fresh-cut products are two-fold. Firstly, fresh-cut products are

highly perishable because mechanical processes such as cutting,

slicing, shredding, and trimming disrupt cellular structures (Rolle and

Chism, 1987; Watada et al., 1996). Secondly, mechanical wounding

results in increased production of ethylene, weight loss, and

respiration rates (Watada et al., 1996). In addition, the cellular

breakdown leads to undesirable enzymatic reactions and tissue

softening, leakage of ions and other cellular components, and

consequently storage life is often reduced (Burns, 1995; Luna-

Guzman et al., 1999). If all these changes cannot be properly

controlled, they can lead to rapid senescence and deterioration of the

product.

It is known that fresh-cut fruit are more difficult to produce than

fresh- cut vegetables. Part of the reason for this difference is that

many fruits have different maturity stages and must be ripened before

they are processed (Cantwell and Suslow, 2002). Commercially,

tomato is a vegetable type of product, but botanically tomato is a

fleshy fruit that undergoes ripening during development. Ripening in

tomatoes is distinct and dramatic (Madhavi and Salunkhe, 1998).

Fresh-cut tomato slices have a relatively short storage life (Artes et al.,

1999; Hong and Gross, 1998), due to the inherent perishability of

tomato and to the cellular damage caused by the slicing process

which leads to tissue softening and associated loss of tissue integrity.

These factors can result in tomato slices with poor quality. Study of

the physiology, biochemistry and nutritional changes of fresh-cut

tomato slices is needed to ensure high quality of sliced tomato

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products during storage, and to provide a scientific basis for

developing effective handling procedures for such a delicate product.

This thesis is divided into 10 Chapters. In Chapter 2, the literature

review will discuss the postharvest physiology of intact and sliced

tomatoes, and give emphasis to the differences that slicing imposes.

The objectives of the study are detailed at the end of literature review.

Chapter 3 covers the general methods applied in all experiments to

prevent repetition in the subsequent chapters. Chapters 4 through 9

present results of the experiments. The final chapter (Chapter 10)

contains the overall discussion and conclusions.

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2. LITERATURE REVIEW

2.1 Tomato Biology

2.1.1 Introduction

The tomato (Lycopersicon esculentum L.) belongs to the family

Solanaceae and is an important horticultural crop. The plant is

indigenous to South America and was domesticated and cultivated in

Mexico but was introduced to Europe and North America in the early

19th century. The popularity of tomato led to extensive breeding

programs to produce cultivars suitable for fresh and processed

consumption (Tigchelaar, 1986; Varga and Bruinsma, 1986). The

tomato is now one of the most widely grown and consumed vegetables

in the world (Rubatzky and Yamaguchi, 1997).

In Australia, tomatoes are gaining in popularity, as annual

consumption per capita has risen from 15 kg to 17 kg during the five

year period 1990 to 1995 (Ross, 1998). Queensland is a leading state

for vegetable production and mainly produces fresh market tomatoes

with only a small percentage of the total crop grown for processing.

The main Queensland growing areas are Bowen, Bundaberg, and

southeast Queensland (Redlands, Lockyer Valley and the Granite Belt)

(Fullelove et al., 1998).

2.1.2 Fruit structure

Tomatoes follow the pattern of growth of parthenocarpic fruit i.e. the

growth of a fruit without embryo development (Varga and Bruinsma,

1986). Botanically, the tomato is a fleshy fruit, specifically a berry,

since the seeds are formed within a fleshy mesocarp (Davies and

Hobson, 1981). Generally, tomato fruit consists of flesh

(skin/epidermis and pericarp) as well as pulp (placenta and locular

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contents) (Ho and Hewitt, 1986). Figure 2.1 depicts the transverse

section of a mature tomato fruit showing the various structures and

regions.

Figure 2.1 The transverse section of a mature tomato fruit (Brecht, 1987)

The skin. The skin or peel of the pericarp consists of the epidermal

layer and 3-5 layers of thick-walled collenchymous tissue (Davies and

Hobson, 1981; Varga and Bruinsma, 1986). The outer epidermis cells

do not have any stomata (Varga and Bruinsma, 1986).

The pericarp. The pericarp consists of an outer pericarp, radial

pericarp and inner pericarp/columella (Ho and Hewitt, 1986). As the

fruit matures, the pericarp continues to enlarge by cell division and

cell enlargement, then the pericarp cells become large with many

intercellular spaces (Davies and Hobson, 1981; Varga and Bruinsma,

1986). The structure of the radial pericarp and inner pericarp is

similar to that of the outer pericarp. The columella has larger air

spaces (Ho and Hewitt, 1986), and contains many starch grains until

the fruit begin to colour (Varga and Bruinsma, 1986). Salunkhe et al.

(1974) indicated that outer and inner pericarp regions play an

important role in the quality of the tomato because of the highest

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contents of dry matter, insoluble solids, and reducing sugars are

found there.

The locules. The locular section contains the seeds that are

surrounded by a gel of parenchyma cells (Madhavi and Salunkhe,

1998). Brecht (1987) reported that the locular or juicy tissue of the

tomato was derived from the placenta of the fruit, overgrowing the

seeds and filling the loculus. However, these are not fused with either

the seeds or the pericarp. Sawhney and Dabbs (1978) found that

increasing locule number does not necessarily result in an increase in

seed number. They also concluded that both locule number and seed

number may independently affect final fruit size.

Shape and Size. The ultimate fruit shape and size are determined by

the rate and duration of cell enlargement (Ho, 1992), cultivar (Bertin

et al., 1998), and environmental and nutritional conditions (Barrett et

al., 1998). Tomato shape varies greatly with cultivar, and can be

elongated or pear like, oblate, or spherical (Barrett et al., 1998).

2.1.3 Fruit composition Tomato fruit consists of 93 - 95% of water (Davies and Hobson, 1981;

Wills, 1987). Generally, 5 - 7.5% of tomato content is dry matter

(Davies and Hobson, 1981) with approximately 1% in cuticles and

seeds, and 4 to 6% in soluble solids (Petro-Turza, 1986). The main

reducing sugars are glucose and fructose with the fructose content

being slightly higher (Baldwin et al., 1991a; Baldwin et al., 1991b;

Stevens et al., 1977b; Young et al., 1993). Sucrose has also been

found in tomatoes, but at much lower concentrations (Davies and

Hobson, 1981; Picha, 1986). Citric acid is the predominant organic

acid in tomatoes but malic acid is also present as a major constituent,

and together they account for 12% of the dry matter (Knee and Finger,

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1992; Stevens, 1985). Figure 2.2 shows the dry matter composition of

the tomato.

Figure 2.2 Composition of dry matter in tomato fruits (Stevens, 1985)

2.2 Fruit quality

2.2.1 Introduction It is generally accepted that consumer satisfaction is associated with

product quality. Quality can be defined as "the sum total of those

attributes which combine to make fruits and vegetables acceptable,

desirable, and nutritionally valuable as human food" (Salunkhe et al.,

1991a). Many preharvest and postharvest factors determine tomato

quality. Quality components that are important for tomatoes are both

external (colour and texture) and internal (flavour and nutritive value)

(Table 2.1). These quality parameters are related to fruit composition

at harvest and they change during postharvest handling.

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Table 2.1 Quality components of tomato

Main factors Components Appearance (visual)

Size: dimensions, weight, volume Shape Colour Defects: physical, physiological, pathological

Texture (feel) Firmness, softness Flavour (taste, smell)

Sweetness Sourness (acidity) Aroma (volatile compounds) Off-flavours and off-doors

Nutritive value Carbohydrates Proteins Lipids Vitamins

Adapted from Salunkhe et al. (1991a).

The following section of this literature review will discuss colour,

firmness, flavour, and nutritional value.

2.2.2 Colour Colour has a strong influence on consumer perceptions (D'Souza et

al., 1992; Francis, 1980) and is an important quality attribute in the

tomato industry (Gould, 1992; Stevens and Rick, 1986). Tijskens and

Evelo (1994) stated that colour is often used as an external index for

overall quality. Consumers prefer tomatoes that are harvested ‘red

ripe’ (Kader et al., 1977; Watada and Aulenbach, 1979).

Shewfelt et al. (1988) proposed that measurement of colour is closely

related to visual perception in tomatoes. Fruit surface colour can be

evaluated by a tristimulus colorimeter, which is known as the Hunter

method. This instrument provides 3 values for each colour

measurement. These values are derived from 3 scales defined as L*,

a*, b* (CIELAB) (McGuire, 1992). "L*" represents brightness/lightness

on a scale 0 - 100, with 0 being perfect black and 100 being perfect

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white. On the horizontal axis, the "a*" scale progresses from green

(negative values) to red (positive values). On the vertical axis, the “b*”

scale covers the range from blue to yellow. Positive “b*” indicates

yellow and negative “b*” indicates blue. As tomatoes turn from green

to red, changes in the colour space are characterised by lower L

readings, a change from –a* to +a* readings and decreased +b*

readings (Shewfelt et al., 1987).

Little (1975) and McGuire (1992) suggested that a more appropriate

measure of colour can be obtained from the calculation of hue angle

(h°) (Figure 2.3). The hue is the actual colour (for example, red,

yellow, blue, etc.), which is effective for visualising the colour

appearance of food products (Little, 1975; McGuire, 1992). A hue of

180° represents pure green and a hue of 0° pure red (Shewfelt et al.,

1988) and is calculated from the arctangent of b*/a*, designated hº.

Arctangent, however, assumes positive values in the first and third

quadrants and negative values in the second and fourth quadrants.

For a useful interpretation, h° should be a positive value between 0º

and 360º.

Figure 2.3. Hue angle, a* and b* colour (McGuire, 1992)

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Hue angle provides the best objective means of ripeness classification.

Shewfelt et al., (1992) proposed ranges as follows: for ‘mature-green’

(hº > 114º), ‘breaker’ (101º < hº < 114º), ‘turning’ (85º < hº < 101º),

‘pink’ (64º < hº < 85º), ‘light red’ (36º < hº < 64º) and ‘red’ (hº< 36º)

classes using average hue at the circumference. Hue angle at the

blossom end was 2-12° lower than at the circumference due to

initiation of colour development at the blossom end (Shewfelt et al.,

1992).

2.2.3 Texture

The texture of fruit is another major quality factor. It is commonly

agreed that consumer preference is for firm fruit that do not lose juice

during eating or slicing (Frenkel and Jen, 1989). The textural quality

of tomatoes is greatly influenced by flesh firmness, the fruits internal

structure (ratio between amount of pericarp and locular tissue), and

skin toughness (cuticle thickness) (Dorais et al., 2001; Frenkel and

Jen, 1989; Kader et al., 1978a). Moreover, the proportion of these

components affects the overall firmness of the fruit (Frenkel and Jen,

1989). This was shown by Barrett et al. (1998) who proposed that

thicker pericarp walls, more pericarp tissue, and fewer locules

ensured the structural integrity of tomato fruit. Moreover, Wann

(1996) explained that fruit firmness can be controlled by the integrity

of cell wall tissues, the elasticity of pericarp tissues, and the

enzymatic activity involved in fruit softening during the process of

ripening. Fruit softening is discussed further in section 2.4.5.

The texture of the tomato can be measured as firmness. In measuring

tomato slice firmness, Wu and Abbott (2002) suggested that a 4 mm

cylindrical probe provided more consistent firmness measurements

than a 6.4 mm spherical probe at harvest, and penetration depth of 3

mm provided more consistent results than 1 mm. Hall (1987)

24

measured the firmness of outer, radial, and inner pericarp tissues

using a 4.9 mm cylindrical probe on 12.5 mm thick tomato slices, and

stated that the outer pericarp was generally firmer than the columella.

Most of the softening of the tissues had occurred by 4 days after

incipient colour development, and tissue-softening rates of the

different tissues were cultivar dependent.

2.2.4 Sugars

Quality of tomatoes is mainly determined by the amount of soluble

sugars. The soluble solids of tomatoes are predominantly sugars

which constitutes about 55-60% of the dry matter (Fig. 2.2)(Hobson

and Grierson, 1993; Stevens, 1985). These sugars, in turn, are

important contributors to flavour. The free sugars are mainly

D-glucose and D-fructose which are present in approximately equal

amounts (Salunkhe et al., 1974). Fructose contributes more to

sweetness than glucose (Salunkhe et al., 1974; Stevens et al., 1977a).

Davies and Kempton (1975) and DeBruyn et al. (1971) indicated that

soluble solids are higher in the pericarp than the locular tissue.

Lower and Thompson (1966) found soluble solids content to be about

equal between the locular and pericarp portions, but Brecht et al.

(1976) showed that the locular tissue contained more soluble solids

than the pericarp. According to Brecht et al. (1976) the soluble solids

of the whole fruit may be higher, equal to, or less than that of the

pericarp or locular gel, depending on the variety.

Sugar content in tomatoes can be estimated by total soluble solids

and is measured using a refractometer and expressed as % soluble

solids or °Brix. Dry matter content is positively related to the total

sugar content of the fruit or to the ratio of soluble to total solids

(Davies and Hobson, 1981). Most tomato varieties vary in soluble

solids from between 3.2 and 7.0% in ripe tomato fruit (Islam and

25

Khan, 2000; Islam et al., 1996; Jones, 1999; Saltveit and Sharaf,

1992). Refractometers are used to measure the soluble solid contents.

2.2.5 Titratable acidity Titratable acidity is a measure of acid content, more specifically, the

number of protons that can be neutralised. Numerous studies have

indicated that factors such as cultivar (Lower and Thompson, 1966),

and stage of ripeness (Iwahori and Lyons, 1970) influence titratable

acidity values in tomato fruit. In ripe fruit, 40 - 90% of the total acids

are in the form of citric acid (Baldwin et al., 1991a; Baldwin et al.,

1991b). The acidity in ripe tomatoes varies widely between 0.25 and

1.1% citric acid on a fresh weight basis (Baldwin et al., 1991a;

Baldwin et al., 1991b; Young et al., 1993). The acid concentration is

important because it contributes to the flavour or taste of tomato

(Barringer, 2004).

Malic acid is found predominantly in ‘immature-green’ fruit and

decreases during maturation, while citric acid increases (Baldwin et

al., 1991a). Tomato fruit are most acidic when ‘mature-green’ or at

the early ‘breaker’ stage, and then acidity declines during ripening

(Baldwin et al., 1991a; Baldwin et al., 1991b; Knee and Finger, 1992;

Richardson and Hobson, 1987).

Kader et al. (1978b) reported that high quality fruit are characterised

by more than 0.32% titratable acidity (TA) and 3% soluble solids (SS)

and by a SS/TA ratio greater than 10. According to Hobson and

Grierson (1993) the ratio of SS/TA plays a major role in determining

the taste of a tomato, with high sugars and high acids being favoured.

26

2.2.6 Flavour

Flavour is an important quality in tomatoes. The flavour of tomatoes

is a complex sensation based on the taste and aroma of several

volatile compounds (Shewfelt, 1993; Stevens et al., 1977a). Flavour

quality of tomatoes is affected by the concentrations of sugars

(sweetness), organic acids (titratable acidity), phenolic compounds

(astringency), and odor-active volatiles (aroma) (Kader, 2002c).

Tomato flavour is also determined by harvest maturity. Maul et al.

(1998) found higher volatile levels and better sensory scores when

tomatoes were harvested at more mature stages. Kader et al. (1977)

also reported that tomatoes have more 'tomato-like' flavour when

harvested more mature. According to Stevens et al. (1977b) a large

locular portion and high concentration of acids characterise flavourful

cultivars.

Dorais et al. (2001) stated that tomato flavour is related to the balance

between sugars and organic acids (sugars/acids ratio) in the fruit, and

total sugar or acid content. The ratio between sugar (soluble solids)

and acid (titratable acidity) of the tomato fruit is a significant factor in

tomato flavour (Jones, 1999) and frequently used to rate the taste of

tomatoes (Barringer, 2004). However Stevens et al. (1977a)

recommend against this because with the same ratio, tomatoes with a

higher concentration of both sugars and acids taste better than those

with low concentrations of both. Loss in flavour during and after

processing of tomato is especially of concern in fresh-cuts, but limited

research has been done in this area.

2.2.7 Nutritional value Tomatoes are important for human health and well-being.

Nutritionally, tomatoes are rich sources of vitamins, especially

ascorbic acid and β-carotene, and antioxidants such as pro-vitamin A

27

(lycopene) (Rubatzky and Yamaguchi, 1997). The stability of nutritive

value during storage is an important factor to be considered for fresh-

cut tomato slices.

Tomato fruit contains important natural antioxidant substances such

as vitamin C (ascorbic acid) and lycopene. Generally, ascorbic acid

concentration in plant material decreases with maturity, but total

ascorbic acid per plant or fruit increases (Klein, 1987). Moreover, the

ascorbic acid content of raw tomato fruit differs according to cultivar

type. Brecht et al. (1976) showed that the ascorbic acid content of

eight cultivars of ripened tomatoes was 12.5 to 22.5 mg 100 g-1, while

Watada et al. (1976) reported that in 11 cultivars of ripe tomatoes, the

range was 13.7 to 31.8 mg 100 g-1.

There are two main carotenoids in tomato: lycopene (ψ,ψ-carotene)

which is the major carotenoid, and β-carotene (Arias et al., 2000a;

Gould, 1992). As tomatoes develop from ‘immature-green’ to ripe, the

increase in carotenoid content is related to the increase in lycopene

content within the plastids (Fraser et al., 1994; Thompson et al.,

2000). Lycopene, the main red pigment, constitutes 50-83% of the

total pigment content in the ripe tomato fruit (Abushita et al., 1997;

Davies and Hobson, 1981; Fraser et al., 1994). With the onset of

ripening, the lycopene content increases (Davies and Hobson, 1981).

Lycopene concentrations were reported to increase from 0.25 mg kg–1

in green tomatoes to values greater than 40 mg kg–1 in fully ripe fruits

(Thompson et al., 2000). Davies and Hobson (1981) found that

lycopene contents may range between 55 and 80 mg kg–1. Sharma

and Rick (1996) showed that the tomato skin contains about five

times more lycopene (540 mg kg–1; fresh weight basis) than the whole

tomato pulp (110 mg kg–1). Lycopene is also more concentrated in the

outer pericarp than in the locular tissue (McGlasson, 1993). Lycopene

concentration in tomatoes can be determined accurately in the

laboratory by spectophotometric measurements of tissue extracts

28

obtained with acetone (Mencarelli and Saltveit, 1988; Shi and Maguer,

2000).

The second main carotenoid is β-carotene that is about 3 to 7% of the

total carotenoid content (Gould, 1992). According to Fraser et al.

(1994) the maximum level of β-carotene occurs at the breaker or light

red stages, and is more concentrated in the locular tissue than in the

outer pericarp (McGlasson, 1993). Dorais et al. (2001) stated that

pigment concentration in the fruit depends on the cultivar and

growing conditions.

2.2.8 Summary Tomato fruit quality components for both intact tomatoes and tomato

slices include colour, firmness, soluble solids, flavour and nutritional

value, in addition to size, shape and freedom from defects. These

quality attributes cannot be improved after harvest but must be

maintained during storage of tomatoes or tomato slices. Future

research should seek to maintain optimal levels of quality in intact

fruits, as quality of fresh-cut tomato slices is influenced by the quality

of corresponding intact product.

2.3 Factors affecting tomato quality

2.3.1 Introduction

The composition and quality of tomato fruit as well as the

physiological, biochemical and storage stability of fresh-cut products

is influenced by preharvest factors such as cultural practices,

cultivars, and harvest maturity. Storage temperature and relative

humidity are the critical postharvest factors in achieving maximum

storage life of products. This section will focus on the effect of

29

cultural practices, cultivars, harvest maturity, storage temperature,

relative humidity, and chilling injury on the quality of intact and

fresh-cut tomatoes.

2.3.2 Cultural practices Among agronomic factors affecting tomato composition and flavour

are water availability, soil fertility and potassium (Stevens, 1985).

Stevens (1985) indicated that careful water management could result

in an increase in fruit solids. In addition, he argued that there is a

positive relationship between nitrogen availability and soluble solids

content. Addition of potassium fertilisers increased the acid content

of fruits (Stevens, 1985). Only one publication exists on the

agronomic factors for fresh-cut tomato slices. Hong et al. (2000)

conducted experiments to compare changes in the quality of slices of

red tomato fruit during storage at 5 °C. Plants were grown using

black polyethylene or hairy vetch mulches. They reported that

tomatoes from plants grown using hairy vetch mulch may be more

suitable for fresh-cut slices than those grown using black polyethylene

mulch on the basis of quality parameters including firmness, soluble

solids, and titratable acidity (Hong et al., 2000). This indicates that

further information on agronomic factors or cultural practices are

needed to produce high quality fresh-cut tomato slices.

2.3.3 Cultivars

Fresh-cut products are more perishable than the intact fresh product

(Kader, 2002c; Watada et al., 1996), therefore it is imperative to grow

varieties with enhanced shelf life characteristics. Genetic factors have

a direct influence on the quality of tomato fruit. As a consequence,

cultivar selection is of primary importance in determining the final

quality of a tomato fruit. Cultivars vary greatly in selectable attributes

such as colour, flavour, firmness, pest and disease resistance, and

30

skin colour (Madhavi and Salunkhe, 1998; Romig, 1995). Sams

(1999) stated that fruit quality traits may be modified by

environmental factors, but that the genetic background of the plant is

the major factor controlling quality.

There are differences in quality between varieties with extended shelf

life, such as those that ripen slowly and conventional ripening

cultivars. Generally, long shelf life cultivars have less flavour than

traditional cultivars (Jones, 1986). The long shelf life cultivar

‘Daniella’ has been introduced in Australia recently (Fullelove et al.,

1998), and while this was initially welcomed by the processing

industry (Ho, 1999), consumers have complained that the fruit was

not firm or juicy when cut into slices.

Ideally, tomatoes intended for fresh-cut production require similar

cultural practices to tomatoes intended for fresh market production

and need no unique harvesting or postharvest requirements

(Ahvenainen, 1996). The main criteria in assessing the suitability of

tomato cultivars for fresh-cut processing are as follows:

1. Multi-locular structure (Fullelove et al., 1998; Ross, 1998).

2. Low sensitivity to physiological disorders and microbial diseases

(Varoquaux and Mazollier, 2002).

3. Resistance to disease and resistance to mechanical injury

(Varoquaux and Mazollier, 2002).

4. Resistance to elevated carbon dioxide concentration and/or low

oxygen concentration (Varoquaux and Mazollier, 2002).

5. Reduced activity of endogenous fruit softening enzymes, which

contribute to degradative processes (Brecht, 1995; Romig, 1995).

6. Reduced chilling sensitivity that will allow more flexibility in

temperature management and result in better storage life and

quality (Brecht, 1995).

31

2.3.4 Maturity

It is essential to distinguish between the ‘horticultural maturity’ i.e.

the stage that represents optimal eating quality and ‘physiological

maturity’ i.e. the stage of full maturation that allows storage and

subsequent good eating quality (Watada et al., 1984). Tomatoes are

harvested at physiological maturity (Watada et al., 1984) and allowed

to ripen off the plant. Tomatoes are harvested at various stages of

maturity from ‘immature-green’ to the full-red stage (Table 2.2).

Table 2.2 Maturity and ripeness classes for fresh-market tomatoes. Class USDA

classification Description

Immature Mature-green A Mature-green B Mature-green C Breaker Turning Pink Light-red Red Full-red

- 1 1 1 2 3 4 5 6 -

Seed cut by a sharp knife on slicing the fruit; no jellylike material in any of the locules; fruit is more than 10 days from breaker stage Seed fully developed and not cut on slicing fruit; jellylike material in at least one locule; fruit is 6 to 10 days from breaker stage; minimum harvest maturity Jellylike material well developed in locules but fruit still completely green; fruit is 2 to 5 days from breaker stage Internal red coloration at the blossom end, but no external colour change; fruit is 1 to 2 days from breaker stage First external pink or yellow colour at the blossom end More than 10% but not more than 30% of the surface in the aggregate; shows a definite change in colour from green to tannish-yellow, pink, red, or a combination thereof More than 30% but no more than 60% of the surface, in the aggregate, shows pink or red colour More than 60% of the surface, in the aggregate, shows pinkish-red or red, but less than 90% of the surface shows red colour More than 90% of the surface, in the aggregate, shows red colour Fruit has developed full final red colour; fruit is more aromatic and softer than at the red stage

Source: Cantwell and Kasmire (2002).

32

It is essential to use mature fruit with an acceptable eating quality for

processing. Over-mature fruit will deteriorate rapidly. Mallik and

Bhattacharya (1996) found that the shelf life of tomatoes picked at the

‘mature-green’ stage was longer than in those picked at the breaker

stage, however they also found that quality after storage was poor.

Several researchers (Kader et al., 1978b; Kader et al., 1977; Shewfelt,

1990a) have found that fruit harvested at earlier stages of maturity

and room-ripened are less sweet, more sour, less tomato-like, and had

more off-flavour than those harvested at the full-red colour stage. In

contrast, tomato fruit harvested at the turning-red stage were sweeter,

less sour and more tomato-like, with less off-flavour than earlier-

harvested fruit (Kader et al., 1977).

There is a general belief that tomatoes ripened on the vine are of

better quality than tomatoes ripened off the vine (Bisogni et al., 1976;

Kavanagh et al., 1986; Picha, 1986; Stevens, 1986). Bisogni et al.

(1976) pointed out that the ratio of soluble solids to titratable acidity

is higher in tomato fruits ripened on the plant compared to room-

ripened fruit. Moreover, Watada and Aulenbach (1979) found that

vine-ripe fruit were considered sweeter than fruit harvested at

‘mature-green’ and ‘breaker’ stages by sensory panels despite there

being no significant differences in soluble solids content or dry matter.

Arias et al. (2000b) showed that the tomatoes ripened on the vine were

more red and darker than the off-vine-ripened tomatoes. The a*/b*

ratio, the lycopene content, soluble solids, total solids, and firmness

were also higher for the vine-ripened tomatoes (Arias et al., 2000b).

Selecting the optimum harvest maturity of the individual variety for

fresh-cut production is another critical factor that affects eating

quality and storage life (Gorny et al., 1998; Salunkhe and Desai, 1988;

Shewfelt, 1990b; Watada and Qi, 1999b). Artes et al. (1999) reported

that the shelf life of tomato slices could be maintained for 10 days at

33

2 °C for the slow ripening tomato cv. ‘Durinta’ with a long storage life

when harvested at the pink stage of maturity (h° values between 65

and 75°). Mencarelli and Saltveit (1988) reported that slices of the

‘mature-green’ tomato cv. ‘Castlemart’ ripened in the same pattern

when compared with whole ‘mature-green’ fruit. It was concluded

that tomato fruit slices could be ripened to an acceptable level of

quality.

2.3.5 Low temperature In harvested products, temperature has a tremendous effect on the

rate of metabolic processes such as respiration. Brecht (1995) found

that metabolic reactions in fruit and vegetables are reduced 2 - 3

times for each 10 °C reduction in temperature. However, the Q10 (the

ratio of the respiration rates for a 10 °C interval) of various fresh-cut

products varies. Zucchini, tomatoes and kiwifruit have a Q10 of about

3.5, while bell peppers, muskmelon have values of about 8.3 (Watada

et al., 1996). Therefore it is crucial to reduce the temperature in order

to prolong shelf life (Paull, 1999).

Low temperatures are effective in minimising a number of adverse

effects of wounding (Watada et al., 1996), inhibiting respiration,

reducing water loss from plant tissue (Shewfelt, 1986), reducing

overall metabolic activity, inhibiting microbial growth (Brackett, 1987),

and reducing changes in texture, nutrition, aroma and flavour (Paull,

1999).

The optimum storage temperature for storage of tomatoes varies

according to the cultivar, and ripeness of the fruit (Agar et al., 1994;

Mallik and Bhattacharya, 1996). Jones (1999) proposed the optimum

storage temperature for ‘mature-green’ fruit as 12.7 to 15.5 °C for

several days without significant quality loss. At these temperatures,

chilling damage does not occur, but colour development is slow. An

34

optimum storage temperature for ripe fruit to prevent significant

quality loss is 7.2 to 10 °C with a relative humidity of 85 to 96%

(Jones, 1999). This implies that red fruit can tolerate storage at lower

temperatures than ‘mature-green’ fruit, as fruit at the advanced stage

(breaker, or turning stage) are less sensitive to chilling injury than

‘mature-green’ fruits (Madhavi and Salunkhe, 1998).

High quality can be assured by maintaining fresh-cut commodities at

lower temperatures, than those recommended for intact fruit and

vegetables (Senesi et al., 2000; Watada and Qi, 1999b). However,

optimal temperatures for fresh-cut product needs to be chosen

adequately in order to avoid damage caused by chilling injury.

Generally, the recommended storage temperature for fresh-cut

vegetables is in the range 0-8 °C (Ahvenainen, 1996; Varoquaux and

Wiley, 1994). Temperature of 0 °C is in most cases preferable,

however it is not economically achievable. Temperatures between 5

and 10 °C are more commonly found in practice (Verlinden and

Nicolai, 2000). Artes et al. (1999) observed significant differences in

quality attributes of fresh-cut tomato on the basis of temperatures.

They found that when compared to 10 °C, tomato slices kept at 2 °C

had better visual quality. Moreover, Gil et al. (2002) considered 5 °C

is the optimum storage temperature to prevent chilling injury and

promote maximum shelf life. In most experiments on tomato slices,

Hong and Gross (1998; 2000; 2001) used 5 °C for storage of fresh-cut

tomato slices.

2.3.6 Relative humidity The relative humidity during storage is another decisive environmental

factor as it directly affects water loss. Water loss causes weight loss

resulting in dehydration and deterioration of tomato fruit as well as

reducing their commercial value (Kays, 1991; Wills et al., 1998).

35

Water (weight) loss from a fruit primarily depends on the water vapour

pressure deficit between the fruit and the storage atmosphere and the

magnitude of resistance to water vapour movement between the fruit

and the air (Rajapakse, 2001; Wills et al., 1998).

Fresh-cut products have exposed internal tissues as well as a large

surface area without any skin. Thus, this product is highly

susceptible to water loss, particularly at higher temperatures where

the vapour pressure deficit is large (Watada et al., 1996; Watada and

Qi, 1999a). However, Watada and Qi (1999a) mentioned that low

relative humidity is not a problem in fresh-cut products, since they

are packaged in film bags or containers over-wrapped with film.

2.3.7 Chilling injury The quality and postharvest storage life of tomatoes can be limited by

chilling injury. Chilling injury refers to an irreversible physiological

disorder observed in plant tissue that results from the exposure of

chilling-sensitive plants or fruits to temperatures below some critical

threshold (Lyons and Breidenbach, 1987). Tomatoes, depending on

maturity, are highly sensitive to chilling injury at temperatures below

12 to 13 °C (Cote et al., 1993; Hobson, 1987). Izumi and Watada

(1995) suggested that for storage of some chilling sensitive

commodities, including tomatoes, 5 °C is preferred to 0 °C.

Raison and Orr (1990) indicated that chilling injury develops in two

stages, referred to as the primary and secondary events. The primary

events are initiated when the produce is stored below the critical

temperatures that cause metabolic dysfunction leading to internal

damage in the cells. The secondary events are a consequence of the

primary events and lead to cell death and visible symptoms (Raison

and Orr, 1990). Based on the observations of chilling-induced lipid

36

degradation in cucumber fruit, Parkin and Kuo (1989) showed that

lipid peroxidation may be associated with chilling injury.

A theory of the nature of chilling injury in plants was explained by

Lyons (1973) (Fig. 2.4). Chilling injury is considered to be a

consequence of bulk lipid-phase membrane transitions occurring at a

critical temperature that leads to a complete loss of permeability

control. Finally, an irreversible metabolic imbalance arises leading to

physiological dysfunction and tissue death (Lyons, 1973).

Figure 2.4 Schematic pathway of the events leading to chilling injury in sensitive plant tissue (Lyons, 1973).

Chilling causes cell membrane damage and increases solute or

electrolyte leakage (King and Ludford, 1983). The electrolyte leakage

is generally considered an indirect measure of plant cell membrane

damage (King and Ludford, 1983; Wang, 1989). The extent of chilling

injury can therefore be determined by measuring the electrical

conductivity of solutes that have leaked from tissues (Autio and

Bramlage, 1986; Murata and Tatsumi, 1979).

37

The disorders induced by chilling temperature usually become

apparent only upon returning the fruit to ambient temperature. The

main symptoms are uneven or partial ripening, fruit softening,

enhanced susceptibility to postharvest fungal pathogens, water

soaked areas and surface pitting (Hobson, 1987; King and Ludford,

1983). In tomatoes, Hobson (1987) used the extent of visible damage,

i.e. water-soaked areas and surface pitting, as an indicator of the

degree of chilling injury. Hong and Gross (2000) found that the

formation of water-soaked areas often occurs while tomato slices are

held in cold storage, and before removal of fruit to non-chilling

temperatures.

One distinct type of tomato chilling injury is the loss of aroma

compounds. Kader et al. (1978b) showed that chilling tomato fruit to

5 °C for one week with subsequent ripening at 20 °C reduced fruit

flavour. In other studies, Maul et al. (2000) demonstrated that

tomatoes stored at 2, 5, 10 or 12.5 °C had less aroma and ripe-

flavours as well as more off-flavours compared with fruit stored at

20 °C. Lurie and Klein (1992b) showed that chill-injured tomatoes

ripened abnormally or they lacked the ability to fully ripen. This was

associated with injury to membranes (Saltveit, 1989).

The most effective method for preventing chilling injury symptoms in

tomatoes is by avoiding exposure of fruit to temperatures below the

critical temperature (Salunkhe et al., 1991a). Madhavi and Salunkhe

(1998) suggested that holding tomatoes below 10 °C for more than 24

hours should be avoided because chilling injury might seriously affect

the market quality of the fruit. Hong and Gross (2000) demonstrated

that the accumulation of ethylene around tomato slices at the ‘light-

red’ maturity stage in containers inhibited the development of chilling

injury.

38

2.3.8 Summary The effects of preharvest (cultural practices and harvest maturity) and

postharvest (storage temperature, humidity, and chilling injury)

conditions have major impacts on the quality of intact tomatoes and

tomato slices. Selection of appropriate tomato cultivars for slicing is

an important breeding objective. Postharvest stress, including chilling

injury, often leads to products deterioration. Therefore by controlling

factors that have a deteriorative effect on the quality of tomato slices

during storage, it is possible to attain good quality tomato slices with

sufficient storage life. Further research should focus in manipulating

the preharvest and postharvest factors for optimal quality of tomato

slices.

2.4 Physiological changes during fruit ripening

2.4.1 Introduction

Ripening is the terminal phase in the development of fleshy fruits and

a process of highly coordinated synthetic and degradative reactions

(Kays, 1991; Rhodes, 1980). During senescence the balance in

dynamic processes shifts with the total degradative reactions

becoming greater than the total synthetic reactions (Watada et al.,

1990). During the ripening of the tomato, from the ‘mature-green’ to

the ‘fully-ripe’ state, the colour, flavour, aroma, texture of tomato fruit

changes dramatically (Grierson and Kader, 1986; Kinet and Peet,

1997), but ripening also initiates fruit senescence and deterioration

(Romani, 1984).

Ripening in tomatoes starts in the interior fruit tissue with gel

formation in the locule. It proceeds to the placenta tissue, columella,

radial pericarp, and eventually the outer pericarp (Brecht, 1987).

Davies and Hobson (1981) stated that tomatoes typically ripen from

39

the "inside-out" and internal colour development and tissue softening

precedes changes in external colour and firmness. Ripening also

progresses from the central columella region down to the blossom end

and then up to the stem-end. Figure 2.5 depicts changes in

metabolism and composition during tomato ripening.

Figure 2.5 Changes in metabolism and composition during ripening (Grierson and Kader, 1986).

The main physiological changes in tomato relate to ethylene

biosynthesis, respiration rates, colour change, fruit softening,

degradation of starch and changes in sugar composition, and tissue

permeability.

2.4.2 Ethylene biosynthesis and production Ethylene is a plant hormone that regulates many aspects of plant

growth, development and senescence (Abeles et al., 1992a; Yang and

Hoffman, 1984). Ethylene is synthesized in, and evolved from, cells of

all climacteric fruits during their growth and development (Abeles et

al., 1992a). It is commonly said that ethylene is “the ripening

40

hormone” (McKeon et al., 1995). It therefore plays an important

regulatory role in the postharvest physiology of horticultural

commodities. The efficiency of postharvest technology systems can be

improved by the ability to control ethylene synthesis and/or responses

to suit specific practical needs (Yang, 1985; Yang and Hoffman, 1984).

Ethylene is formed from methionine by the following steps:

S-adenosylmethionine is synthesised by the action of SAM synthase

on methionine (Fig. 2.6). ACC synthase then converts SAM to

1-aminocyclopropane-1-carboxylic acid (ACC), which is, in turn,

converted enzymatically to ethylene by the action of ACC oxidase

(Yang, 1987; Yang and Hoffman, 1984).

Enzymes catalyse each step in the ethylene biosynthesis pathway.

Two enzymes that are unique to this pathway are ACC synthase and

ACC oxidase (Fig. 2.6) (Imaseki, 1991; Yang and Hoffman, 1984).

Measurements by Su et al. (1984) indicate that ACC synthase activity

increases quite early during ripening of tomatoes, and Liu et al. (1985)

found that ACC oxidase activity is enhanced by exogenous ethylene in

preclimacteric tomatoes.

ACC synthase is the main control site for ethylene biosynthesis. ACC

synthase plays a major role during autocatalysis (positive feedback)

where ACC synthase is stimulated, as well as during auto-inhibition

(negative feedback) where ACC synthase is inhibited (Yang and

Hoffman, 1984). Cameron et al. (1979) have shown that the

application of ACC to various plant organs, including roots, stems,

leaves, inflorescences, and fruit, results in a marked increase in

ethylene production. ACC synthase is strongly inhibited by inhibitors

of pyridoxal phosphate-dependent enzymes such as AVG and AOA

(Yang, 1985).

41

Figure 2.6 Pathway of ethylene biosynthesis (Reid, 2002)

Ethylene moves by diffusion from its site of synthesis (Davies, 1995;

Reid, 1995). The stem-scar of tomatoes serves as the main avenue for

ethylene and CO2 exchange (Burg and Burg, 1965; De Vries et al.,

1995b). This was supported by De Vries et al. (1995a) who showed

that more than 90% of the total ethylene is released through the stem-

scar region after removal of the calyx.

Ethylene synthesis is stimulated in most tissues in response to stress.

In particular, it is synthesised in tissues undergoing senescence or

ripening (Davies, 1995; Picton et al., 1995; Reid, 1995). In addition,

various environmental stresses such as mechanical wounding

(bruising and cutting) (Abeles et al., 1992a) and chilling temperatures

(Wang, 1993) stimulate ethylene formation.

Methionine

ATP

PPi + Pi

SAM synthase

S-adenosylmethionine (SAM)

ACC synthase

1-aminocyclopropane-

O2

CO2 + HCN

ACC oxidase

Ethylene

42

Theologis et al. (1992) considered the function of ethylene as a

“coordinator” of ripening. They established that ethylene regulates

fruit ripening by coordinating the expression of many genes involved

in metabolic processes, such as increasing fruit respiration rates,

chlorophyll degradation, synthesis of carotenoids, conversion of starch

into sugars, and the activity of several enzymes involved in the

degradation of cell walls. Ethylene also stimulates its own production.

In studies with transgenic and mutant tomato lines with inhibited

ethylene biosynthesis or perception, Giovannoni (2001) showed that

the process of climacteric ripening represents a combination of

ethylene regulation and developmental control. He suggested that

both ethylene and additional developmental processes regulate fruit

ripening.

A simple scheme that depicts features of the mechanism of ethylene

action in plants is shown at Figure 2.7. It is believed that ethyene

binds to a molecule, the “receptor” at the cell membrane. At the

binding site an activated complex is formed which stimulates release

of a “second message” that migrates to the cell nucleus and in turn

causes synthesis of mRNA (messenger RNA). The new mRNA

molecules direct the synthesis of polypeptides that form enzymes that

cause ethylene-induced actions, including fruit ripening (Alexander

and Grierson, 2002). The physiological effects of ethylene can be

blocked by ethylene binding inhibitors such as 1-MCP, and this will

be discussed in section 2.6.4.

Sisler and Goren (1981) demonstrated that ethylene binds to a protein

receptor and that such binding is a prerequisite for the manifestation

of hormonal effects. Leshem (1992) stated that the total number of

ethylene binding sites gradually increases with time and peaks when

tissue is still relatively young and declines afterwards, whereas

maximal ethylene production is considerably later (Leshem, 1992).

43

Figure 2.7 Mechanism of ethylene action (Reid and Wu, 1991)

In climacteric fruit, ethylene production is generally very low until the

commencement of ripening. Internal ethylene concentrations during

fruit growth, up to the start of the climacteric respiration rate, are less

than 1µL L-1 (McMurchie et al., 1972). Tomato produces moderate

amounts of ethylene on a fresh weight basis, around 1.0 – 10.0

µL kg1 h-1 (Kader, 2002b) . Brecht (1987) reported that in ‘mature-

green’ fruit the ethylene production rate is 0.08 - 1.5 nL g-1 h-1.

McMurchie et al. (1972) referred to this basic level as ‘system 1’

production. System 1 is the basal low rate of ethylene production

present in preclimacteric fruits or before the onset of ripening

(McMurchie et al., 1972).

Ethylene synthesis begins to increase at the onset of ripening. This

takes place before any external colour change at the blossom-end of

green fruit becomes noticeable and precedes the synthesis of enzymes

44

such as polygalacturonase (Grierson and Tucker, 1983; Su et al.,

1984). According to Chalmers and Rowan (1971) the climacteric peak

in ethylene evolution occurs between the ‘mature-green’ and ‘pre-

breaker’ stages. Although, Lyons and Pratt (1964) reported that the

highest level of ethylene production in tomato fruit occurs at the ‘pink’

stage of ripening.

The rise in ethylene production is an autocatalytic process (i.e.

ethylene stimulates its own synthesis) which coincides with a rise in

climacteric respiration (Jeffery et al., 1984). Campbell et al. (1990)

reported that ethylene production reaches a peak of 12 - 15 nL g-1 h-1

on a fresh weight basis. The rate of ethylene production during

maturation and ripening correlates well with endogenous levels of

ACC, ACC synthase and ACC oxidase (Hoffman and Yang, 1980).

Hobson and Grierson (1993) introduced the concept of ‘system 2

evolution’ to represents the high rate of autocatalytic ethylene

production accompanying ripening in climacteric fruit (Oetiker and

Yang, 1995).

2.4.3 Respiration Fruit have been classified as climacteric or non-climacteric based on

their respiratory and ethylene production patterns during ripening

and their response to exogenous ethylene (Biale and Young, 1981;

Kader, 2002b). The tomato has been classified as a climacteric fruit

(Kinet and Peet, 1997). Early in development, the respiration rate is

high and decreases to a pre-climacteric minimum during maturation.

At the onset of ripening, respiration increases to a maximum, the

climacteric peak, before it subsequently declines slowly (Biale and

Young, 1981). This respiratory peak is preceded by or associated with

a rise in ethylene production (Kinet and Peet, 1997; Wills et al., 1998).

At the climacteric peak, respiration rate, measured as CO2 production,

45

is approximately double the pre-climacteric minimum and ranges

from 26 to 50 µL CO2 g-1 h-1 on a fresh weight basis (Andrews, 1995;

Campbell et al., 1990). At the pink-red stage, the climacteric process

of respiration reaches the maximum level (Salunkhe and Desai, 1984).

According to Meir et al. (1992) and Maharaj et al. (1999) respiration

rate is one of the most important indicators of senescence in tomato

fruit.

2.4.4 Loss of chlorophyll and synthesis of lycopene

The principal pigments that are responsible for the colour of tomatoes

are chlorophyll and carotenoids, especially lycopene (Arias et al.,

2000a; Hobson and Davies, 1971). Chlorophyll is the major pigment

in the early stages of tomato fruit development that imparts the green

colour. As fruit mature and ripen, the chlorophyll content decreases

because chloroplasts are converted to chromoplasts and additional

carotenoids are synthesised (D'Souza et al., 1992; Hobson and Davies,

1971). According to Fraser et al. (1994) and Mencarelli and Saltveit

(1988) the chlorophyll content is reduced by 90% by the time

tomatoes are red-ripe.

The intensity of the bright red colour of tomatoes is mainly due to the

presence of lycopene (Johjima and Matsuzoe, 1995; Stevens and Rick,

1986). Lycopene increases as tomato mature (Shi and Maguer, 2000).

Dumas et al. (2002) mentioned that at lycopene concentrations (on a

fresh weight basis) between 32 and 43 mg kg–1, fruit colour turns from

orange to red. Moreover, they state that lycopene accounts for 90% of

total carotene at red colouration (Dumas et al., 2002).

After harvest, colour development of tomatoes during ripening is

influenced by many factors including temperature (Grierson and

Kader, 1986). Shewfelt et al. (1988), Tijskens and Evelo (1994), and

46

Dumas et al. (2002) proposed a relationship between temperature and

the presence of lycopene. They indicated that at normal temperatures

(12 to 25 °C) chlorophyll degrades, while lycopene, and to a minor

extent, β-carotene is formed, resulting in red-coloured tomatoes. At

extreme temperatures such as temperatures below 12 °C, chlorophyll

does not degrade and lycopene does not accumulate. Prolonged

chilling also leads to loss in the ability to ripen even at normal

ripening temperatures, resulting in yellowish-green tomatoes. At high

temperatures, above 30 °C, chlorophyll degrades and β-carotene

accumulates, but the synthesis of lycopene is inhibited, resulting in

orange-yellow fruit.

2.4.5 Fruit softening Softening is generally associated with the ripening of fleshy fruits.

Flesh softening during fruit ripening is dependent on changes in the

chemical structure of the cell wall components (Kojima et al., 1991;

Tucker, 1993), protein, and the 3 carbohydrate fractions of pectin,

cellulose, and hemicellulose (John and Dey, 1986). Fruit softening is

characterised by increases in soluble pectins (John and Dey, 1986).

Pectin is a major component of the middle lamella, which binds

adjacent cells. A textural change as a result of solubilization of pectin

during ripening has been demonstrated in tomatoes (Gross and

Wallner, 1979; Hobson and Davies, 1971).

Fruit softening is stimulated by enzymatic and non-enzymatic

processes during ripening (Gross, 1990). The production of enzymes

involved in cell wall degradation is greatly accelerated during tomato

ripening (Poovaiah et al., 1988). Enzymes capable of degrading pectin

in fruits include pectinmethylesterase and polygalacturonase (Gross,

1990; John and Dey, 1986; Rigney and Wills, 1981).

47

Pectinmethylesterase catalyses the hydrolysis of methyl esters in

pectin molecules (Kays, 1991). Pectinmethylesterase activity in

tomato occurs throughout fruit development and ripening (Tucker et

al., 1982). Hobson and Grierson (1993) indicated that

pectinmethylesterase may be implicated in softening initiation. They

also stated that pectinmethylesterase and polygalacturonase occupy

different sites in the cell wall and middle lamella.

Polygalacturonase plays a role in the dissolution of the middle lamella

during ripening (John and Dey, 1986). Tucker and Grierson (1982)

reported that polygalacturonase activity was absent in ‘green’ tomato

fruit, but was associated more with softening in the later stages of

ripening (Gross, 1990). Tieman and Handa (1989) demonstrated that

polygalacturonase first appeared in the columella region (Figure 2.1)

followed by sequential appearance in the exopericarp and

endopericarp, respectively. These results suggest a regional

degradation of pectic substances in the fruits by polygalacturonase

(Kojima et al., 1991). However, the precise role of polygalacturonase

in softening of tomatoes is not yet clear. Smith et al. (1988) found

that if transgenic tomato plants were produced in which the level of

polygalacturonase synthesis in the fruit was substantially reduced

(about 90%), fruit softening was not significantly different from normal

non-transformed fruit.

2.4.6 Degradation of starch, and sugar changes Sugars originate from photosynthetic assimilates. Tomato fruit

accumulates carbohydrate prior to the onset of ripening in the form of

starch (Tucker, 1993). Madhavi and Salunkhe (1998) have noted that

tomato fruits accumulated low levels of starch in the immature stages.

Starch accumulation continues up to the ‘mature-green’ stage and

then rapidly decreases as ripening begin (Yu et al., 1967). Hobson

and Davies (1971) found that starch constitutes 0.10 - 0.15% in ripe

48

tomato fruit on a dry weight basis, and was hydrolysed during

ripening.

The breakdown of starch to glucose, fructose or sucrose is associated

with activities of α- and β-amylases and starch phosphorylase (Presis

and Levi, 1980; Steup, 1988). Generally, the sugar content of tomato

fruit is a function of the stage of maturity (Salunkhe et al., 1974).

According to Richardson and Hobson (1987) and Baldwin et al.

(1991a) sugar content increases progressively from the ‘mature-green’

or ‘breaker’ stages to reach the maximum at ‘turning’ or ‘red-ripe’

stages, but decreases once the fruit has begun to colour. Sugars in

the locular gel and the outer pericarp also increase during

development and maturation (Winsor et al., 1962a; Winsor et al.,

1962b).

2.4.7 Changes in cellular membranes Among the mechanisms associated with tomato fruit ripening,

changes in membrane structure play an important role. The cell

membrane system (i.e. plasma membrane, endoplasmic reticulum,

vacuolar membrane etc.) acts as selectively permeable barriers to the

movement of compounds within and between cells. Membrane

structure consists of fluid bilayers containing phospholipids and

proteins (Marangoni et al., 1996; Paliyath and Droillard, 1992;

Stanley, 1991). Generally, there is a decline in membrane

phospholipid content during ripening of tomato fruit (Bergevin et al.,

1993; Nguyen and Mazliak, 1990; Whitaker, 1991). Gucluu et al.

(1989) reported a correlation between changes in lipid composition,

particularly in the sterol-phospholipids ratio, and increased

membrane permeability.

Senescence is characterised by degradation of cell membranes and a

loss of membrane integrity and function, which in turn leads to loss of

49

tissue structure, alterations in cellular metabolism and ultimately

accelerated death (Nooden and Leopold, 1988; Paliyath and Droillard,

1992). One significant change in the membranes occurring with

senescence is the change in fluidity. Thompson et al. (1982) showed

that with the development of senescence, fluidity decreases and the

membranes becomes more rigid. This change can alter the activity of

enzymes that are associated with the membrane, and the function of

receptors on the membrane (Kays, 1991). Another physiological

change is increased membrane permeability, expressed as increasing

leakage of ions (Cote et al., 1993; Palma et al., 1995). With the

assumption that leakage is a result of an increase in membrane

permeability, solute leakage has been used as an indicator of tissue

senescence (Brady et al., 1970). This suggests that decreased fluidity

of membranes is translated into leakage of ions and therefore reduced

functionality of the membranes (Marangoni et al., 1996). Maintaining

cell membrane integrity and functionality is therefore considered as

the physiological basis for the preservation of fresh produce (Lee et al.,

1995).

2.4.8 Summary The ripening of climacteric fruits such as the tomato is stimulated by

ethylene. Ripening has been viewed as a genetically programmed

event involving the regulated expression of specific genes (Grierson,

1987). Dramatic physiological and biochemical changes occur in

tomato fruit during ripening, and those events are associated with

changes in ethylene production, respiratory and enzyme activity,

including cell wall and membrane-associated proteins. The capability

to control tomato ripening by modulating ethylene responses could

extend the storage life of tomatoes, and therefore ethylene production

response to tissue wounding will be reviewed in the next section.

50

2.5 Control of ripening

2.5.1 Introduction

Ethylene is involved in the regulation of many physiological responses.

Ethylene is often used in fruit and vegetable production to achieve

uniform ripening or to initiate ripening (Watada, 1986). The following

review elaborates on ethylene physiology, the role of the anti-ethylene

gas 1-MCP, and physical (heat) treatments for controlling tomato and

tomato slice ripening and quality.

2.5.2 Effect of ethylene on quality attributes The plant hormone ethylene is a gas that is involved in fruit ripening.

Ethylene is effective at parts-per-million (µL L–1) to parts-per-billion

(nL L–1) concentrations (Saltveit, 1999). In banana, Inaba and

Nakamura (1986) maintained that the effect of ethylene treatment

depends on the sensitivity of the fruit, which increases with age. Most

fruit, including tomatoes, become increasingly sensitive to ethylene

with time after anthesis (McGlasson et al., 1978).

The response of plants to ethylene can be beneficial or detrimental.

One of the potentially beneficial responses is induced ripening

(Salunkhe et al., 1991a). Lyons and Pratt (1964) showed exposure to

ethylene accelerates the natural ripening of unripe tomatoes with an

accompanying climacteric rise in CO2 and C2H4 production. The

development of ‘ripe’ colour with ethylene treatment was also observed

in other fruits such as in banana (Liu, 1976) and muskmelon (Bianco

and Pratt, 1977). Detrimental effects can be seen in the work of Risse

and Harton (1982) who showed that exposing watermelons to 5, 30, or

60 µL L–1 C2H4 reduced firmness and rind thickness, accelerated

deterioration, and reduced acceptability after 3 days at 18 °C.

Moreover, cucumbers treated with C2H4 developed unacceptable

51

textural attributes (Poenicke et al., 1977). These confirm that

ethylene has a detrimental effect on the texture of fruits and

vegetables by promoting unwanted softening (Saltveit, 1999).

Biochemical changes in tomato fruits have been divided into three

different classes by Jeffery et al. (1984):

1. Changes in metabolism of starch, sugars and organic acids:

independent of ethylene;

2. Loss of chlorophyll: enhanced by ethylene but does take place in its

absence;

3. Formation of lycopene, and increase in polygalacturonase and

invertase activities: dependent on ethylene for initiation and

continuation of the response.

2.5.3 Control of ethylene action Ethylene action can be manipulated using inhibitors of ethylene and

by removal of ethylene.

Inhibitors of ethylene. The most frequently used inhibitors of ethylene

production in horticulture are AVG (aminoethoxyvinylglycine) and

AOA (aminooxyacetic acid). Both are effective in inhibiting the activity

of ACC synthase (Owens et al., 1971; Yang and Hoffman, 1984).

Baker et al. (1978) found that AVG inhibited ethylene production more

completely in ‘green’ than in ‘pink’ tomato fruit tissues. For example,

ethylene production was inhibited by 69% in ‘mature-green’ tomato

slices, but only by 11 and 13% in climacteric (‘pink’) and post-

climacteric (‘red’) fruit, respectively. Baker et al. (1978) suggested that

this difference in sensitivity to AVG might be explained by increased

competition for methionine by ethylene formation in the riper tissue.

Yang and Hoffman (1984) suggested that because AVG and AOA do

not inhibit the conversion of ACC to ethylene, the level of ACC already

present in the tissues limits their effectiveness.

52

The ethylene inhibitor that has been reported to bind to ethylene

receptors is silver nitrate (AgNO3). However, a problem with using Ag+

is toxicity (Abeles et al., 1992). Another potentially safer inhibitor of

ethylene perception is 1-MCP, which is described below.

Removal of ethylene. The simplest strategy to protect sensitive

produce from exogenous ethylene is to remove ethylene gas from the

environment. Ethylene can be removed from storage areas by

constant ventilation with fresh air (Reid, 2002). Chemically, ethylene

can be removed with compounds that trap or convert it to inactive

products. One such absorbent compound is potassium permanganate

(KMnO4), sold commercially as Purafil® (Reid, 2002).

Potassium permanganate has the ability to oxidise ethylene to form

CO2 and H2O. In this oxidation process, the colour changes from

purple to brown as MnO4- is reduced to MnO2 (Reid, 2002). The use of

KMnO4 absorbents in polyethylene bags has extended the storage life

of bananas (Scott et al., 1970) and avocados (Hatton and Reeder,

1972). However, although the use of KMnO4 is convenient, there are

toxicity problems associated with its safe handling and disposal

(Abeles et al., 1992a).

Ethylene removal will delay fruit softening. Abe and Watada (1991)

maintained that removal of C2H4 from the storage environment of

lightly processed fruit and vegetables can retard tissue softening. Abe

and Watada (1991) used an ethylene absorbent (charcoal with

palladium chloride) to prevent the accumulation of ethylene and found

the absorbent was effective in reducing the rate of softening in peeled

and sliced kiwifruit, and sectioned bananas. Studies by Risse and

Miller (1983) showed that removal of ethylene from the storage

atmosphere increased the number of days for tomatoes to reach full

red colour and they were firmer than tomatoes stored without

ethylene removal. Knee et al. (1985) showed that the use of potassium

53

permanganate can result in reduced softening and /or rotting in

apples, avocado, banana, kiwifruit, lemon and mango.

2.5.4 1-methylcyclopropene The plant growth regulator 1-methylcyclopropene (1-MCP) has been

reported to have inhibitory effects on ethylene action in various

horticultural products such as banana (Jiang et al., 1999a; Macnish

et al., 1998), apple (Fan et al., 1999), broccoli (Fan and Mattheis,

2000b) and lettuce (Fan and Mattheis, 2000a). Application of 1-MCP

has been reported also to fresh-cut products such as apple (Jiang and

Joyce, 2002) and pineapple (Budu and Joyce, 2003).

1-MCP is a gas that has been used on many horticultural products to

delay ripening and senescence (Watkins and Miller, 2003). 1-MCP is

an effective inhibitor of ethylene action at low concentrations (Sisler

and Serek, 1997). Figure 2.8 shows the chemical structure of 1-MCP.

Since 1-MCP is a non-toxic gas and leaves low measurable residues in

food commodities (Watkins, 2002), it can be used as a tool to study its

utility for extending the storage life and quality of plant products

(Blankenship and Dole, 2003).

Figure 2.8 Chemical structure of 1-MCP (Prange and DeLong, 2003)

Generally, 1-MCP has been applied at temperatures ranging from 20 –

25 °C (Blankenship and Dole, 2003; Watkins, 2002). Ku and Wills

(1999) showed that the application of 1-MCP produced better results

54

at 20 °C than at 5 °C. Similarly, 20 °C was best for application of 1-

MCP to broccoli (Able et al., 2002). Blankenship and Dole (2003)

hypothesised that lower temperature might lower the affinity of the

binding site for 1-MCP.

Most literature shows that treatment duration considered sufficient to

achieve a full response to 1-MCP is 12 to 24 hours (Blankenship and

Dole, 2003; Watkins, 2002). Higher concentrations of 1-MCP are

required for shorter treatment times (Ku and Wills, 1999; Wills and

Ku, 2002).

1-MCP is thought to act by binding “for a long time” to the ethylene-

receptor (Sisler and Serek, 1997) so that ethylene cannot elicit

subsequent signal transduction and translation (Sisler and Serek,

1997). The recent theory that 1-MCP may act to block ethylene action

was modelled by Binder and Bleecker (2003). They proposed that 1-

MCP suppresses the ethylene response pathway by permanently

binding to a sufficient number of ethylene receptors (ETR1, ETR2,

EIN4, ERS1 and/or ERS2) that keeps CTR1 in its active (inhibiting)

state (Fig. 2.9).

Figure 2.9 Model of mode of action of 1-MCP proposed by Binder and Bleecker (2003)

Generally, the ripening processes of most climacteric fruit, such as

softening and loss of titratable acidity, are usually delayed or inhibited

by 1-MCP application. 1-MCP delayed softening in intact and cut

apples (Jiang and Joyce, 2002). Hofman et al. (2001) showed that 1-

55

MCP delayed softening in avocado by 4.4 days, custard apple by 3.4

days, and mango by 5.1 days. Titratable acidity loss was inhibited in

tomatoes (Wills and Ku, 2002) and carrots (Fan and Mattheis, 2000a)

by 1-MCP. Application of 1-MCP produced higher soluble solids in

apples (Fan and Mattheis, 1999a), papaya (Hofman et al., 2001) and

pineapples (Selvarajah et al., 2001).

Rohwer and Gladon (2001) demonstrated that tomatoes treated with

1-MCP at the ‘breaker’ and ‘turning’ stages for 8 hours at 20 °C did

not develop any red colour over the 10-day holding period. They

reported that the most desirable delay in ripening was obtained by

treating fruits at the ‘pink’ stage with 0.058 µL L-1 (ca. 3 day delay) or

fruits in the ‘light-red’ stage with 580 nL L-1 1-MCP (ca. 4 day delay).

Mir et al. (1999) applied 1-MCP to tomatoes at 22 °C and reported an

extension of shelf life at all stages of maturity, due to reductions in the

rate of red colour development and loss in firmness.

Postharvest application of 1-MCP is an efficient method for delaying

the ripening of ‘green’ tomatoes and delaying the senescence of ripe

tomatoes. Wills and Ku (2002) reported that exposure of ‘green’

tomatoes to 5 µL L–1 1-MCP for 1 hour resulted in a 70% increase in

time to ripen. They also reported that exposure of ripe tomatoes to 20

µL L–1 for 2 hours resulted in 25% increase in postharvest life, based

on fruit appearance. Furthermore, Moretti et al. (2001) reported that

tomato fruit treated with 1-MCP at 1 µL L–1 for 22 hours at 22 °C were

about 88% firmer than the control fruit, and had a 38% lower a*/b*

ratio (more green colour), than control fruit at the end 17 days

storage.

Little literature exists on the use of 1-MCP on fresh-cut fruit products.

Fresh-cut apples treated with 1-MCP at concentrations of 1 or 10 µL

L–1 for 6 h at 20 °C showed reduced respiration and ethylene

56

production rates and delayed softening (better firmness maintenance)

and colour changes (Jiang and Joyce, 2002). These authors

mentioned that compared with untreated tissue, quality was better

maintained at 4 °C in apple slices treated with 1-MCP before or after

the fruit were processed. In fresh-cut pineapple fruit, Budu and Joyce

(2003) showed that the application of 1-MCP at higher concentrations

(up to 5 µL L–1) reduced the respiration rate and maintained the colour

of the cut pineapple pieces.

Limited information also exists on the efficacy of 1-MCP on fresh-cut

vegetable products. Ku and Wills (1999) showed that broccoli florets

held at low temperature (5 °C) after treatment with 1-MCP from 0.02

to 50 µL L–1 at either 20 or 5 °C had a longer storage life than control

florets. Their data showed that low concentrations of 1-MCP strongly

inhibited the loss of green colour of broccoli florets. The storage life of

shredded lettuce was extended by application of 1-MCP (Wills et al.,

2002), with the optimal treatment being 0.1 µL L–1 for 1 h at 5 °C

which resulted in an extension in storage life by 50% over untreated

lettuce.

2.5.5 Heat treatment

There has been increasing interest in the postharvest heat treatment

(thermotherapy) of vegetables and fruit to control insects pests,

prevent fungal decay, and to modify the ripening of commodities

(Lurie, 1998; McDonald et al., 1999). This is primarily because heat

treatment substitutes as a non-damaging physical treatment. It is a

non-carcinogenic, non-polluting, non-damaging treatment for

prevention of chilling injury and maintenance of fruit and vegetable

quality (Holton, 1990).

57

Several researchers, including Key et al. (1981) and Lurie (1998),

argue that exposure of plant tissues to thermal stress results in the

rapid induction of a small set of specific proteins called heat shock

proteins (HSPs). These proteins are produced within 30 minutes of

exposure to temperatures in the range 34 to 42 °C (Kanabus et al.,

1984). Saltveit (2000) reported that a brief heat shock interferes with

‘normal’ protein synthesis by preferentially inducing the synthesis of a

unique set of stress proteins.

Several authors have obtained prolonged postharvest life using heat

treatments. These include Teitel (1989) with melons, Barrancos et al.

(2003) with apples, and Miller et al. (1991) with mangoes. By

contrast, other authors such as Kerbel et al. (1987) and Chun et al.

(1988) on avocados and grapefruits respectively, have reported

negative effects on fruit quality after the application of heat

treatments. Generally, the main problem in using heat treatment is

the increased weight loss arising from the use of relatively high

temperatures, and the damage related to over-heating, such as

wrinkling or pitting of the fruit skin (Klein and Lurie, 1991; Lurie,

1998).

Methods for heat treatment of harvested fresh fruit and vegetables

include hot water, vapour heat and hot air (Lurie, 1998). Depending

upon exposure duration, if the temperature is too high (≥ 45 °C) or if

the treatment too prolonged, it can be lethal for the fruit (Paull, 1990;

Paull and Chen, 2000). Brief hot water treatments heat only the

surface cells of the product, whereas heated air treatments applied for

several hours increase the pulp temperature significantly (Salunkhe et

al., 1991a).

Tomatoes have been reported to tolerate different exposures to heat

without injury (Lurie and Klein, 1992b). Lurie and Klein (1991) found

that ‘mature-green’ tomatoes held 3 days at 36-40 °C before chilling at

58

2 °C did not develop chilling injury (whereas unheated tomatoes did)

and quality attributes were maintained for 4 weeks.

High temperatures are known to inhibit ethylene production in

tomatoes. Hot air treatment of 35 - 40 °C inhibits ethylene synthesis

within hours (Biggs et al., 1988). These authors correlated this

inhibition with a rapid decline in 1-aminocyclopropane-1-carboxylic

acid synthase activity (Figure 2.6). Yu et al. (1980) showed that heat

treatment blocks, at least transiently, the conversion of ACC to

ethylene. Atta-Aly (1992a) found that during incubation of tomato at

temperatures between 20 and 30 °C, inhibition of ethylene

biosynthesis was attributed to the reduction in ACC synthesis,

whereas at 35 °C, both ACC synthesis and its conversion to ethylene

were inhibited. Some researchers (Klein and Lurie, 1990; Paull and

Chen, 1990) reported a rapid loss (75%) of ACC oxidase in papaya and

other fruits exposed for short periods to temperature greater than

40 °C.

Fruit firmness is also affected by heat treatment. Biggs et al. (1988)

showed that tomatoes softened more slowly when held continuously at

temperatures between 30 °C and 40 °C than at 20 °C. Similarly,

Hinton and Pressey (1980) demonstrated that in the range 35 - 60 °C,

87% loss of activity of purified glucanase from tomato fruit can occur.

This was confirmed by Pressey (1983) who found that 50% glucanase

activity is lost by exposure to 50 °C for 5 minute. Kim et al. (1993a)

showed that heat treatment of whole apples improved apple slice

firmness. In a related experiment, Kim et al. (1994) demonstrated that

slices prepared from heat-treated apples showed increased firmness

during storage for up to 7 days for ‘Golden Delicious’ (firmness 34%

higher than on day zero of storage) and up to 14 days for ‘Delicious’

apple (48% higher firmness than at the beginning of storage).

59

Heating enhances colour development. Lurie and Klein (1992b)

showed that heated (38 °C for 3 days) tomatoes were redder, but not

softer, than non-heated fruit after storage at 12 °C and shelf life at

20 °C. Lycopene synthesis, which is responsible for the development

of red colour, was inhibited in fruits held at temperatures above 30 °C

(Chang et al., 1977). They also showed that when returned to 20 °C,

the inhibition of ripening was reversed and the fruits ripened

normally, although more slowly than fruits kept continually at 20 °C.

Generally, there are variable results on the effect of heat treatments

on soluble solids. This has been shown by several researchers (Lurie

and Klein, 1991; Lurie and Sabehat, 1997; McDonald et al., 1999)

who demonstrated that postharvest heat treatments, either water or

hot air (38 - 48 °C for 1 hour to 3 days), have no effect on tomato

soluble solids or titratable acidity. In addition, Lurie and Klein

(1992b) found that titratable acidity was the same between heated and

non-heated tomatoes, but in this work soluble solids contents

remained higher in heated fruit. They also showed that heating

induced higher sugar/acid ratio by 10 – 30%, which makes it

attractive for consumers. Paull and Chen (2000) maintained that the

variable effects on soluble solids and titratable acidity depends upon

the temperature used and duration.

Most research on the effects of hot-air treatments on tomatoes has

focussed on intact tomatoes at ‘mature-green’ maturity rather than on

‘pink’ or ‘red’ maturity (Klein and Lurie, 1991; Lurie and Klein, 1992b;

Saltveit and Cabrera, 1987). Postharvest heat treatments applied to

fresh-cut commodities have therefore targeted mainly improvement of

post-processing quality rather than shelf life extension such as in

fresh-cut apples by Kim et al. (1994) and Barrancos et al. (2003), in

lettuce by Loaiza-Velarde and Saltveit (2001), in fresh-cut celery

petioles by Loaiza-Velarde et al. (2003), and in fresh-cut cantaloupe by

60

Luna-Guzman et al. (1999). Less information is available on the

effects of high temperatures on tomato slices.

2.5.6 Summary

Control of ripening of tomatoes through the management of ethylene

physiology and its exposure to tomatoes is a vital component in the

postharvest physiology of tomato slices. In order to retard or prevent

quality loss in fresh-cut tomato slices, various possible external

treatments have been described. The inhibitor of endogenous

ethylene action (1-MCP) could be used as a tool to unravel the effects

of ethylene on postharvest quality of tomato slices. Heat treatment is

a promising alternative to chemical treatments, however several

aspects of the application of this technique to fresh-cut tomato slices

requires further research.

2.6 Physiological changes after slicing

2.6.1 Introduction

The physiological changes in fresh-cut products are concerned with

wounded tissue (Brecht, 1995) as the tissue integrity of these

products has been altered during slicing (Rolle and Chism, 1987;

Shewfelt, 1986). Any opening of tissue generally leads to metabolic

changes, so wounding elicits changes in fresh-cut products (Fig. 2.10).

Wounding stress results in metabolic activation including increased

respiration and ethylene production, membrane deterioration and in

some cases, induction of wound-healing processes and enhanced

water loss. Other consequences of wounding include flavour changes

and softening (Brecht, 1995; Saltveit, 1997). Wounding also provides

a suitable media for the growth of micro-organisms with visible

61

disease symptoms while the increased respiratory activity contributes

to a loss of nutritional value (Reyes and Gould, 1996).

Figure 2.10 The interrelationship among the many effects of slicing on the physiological process in fresh-cut products (Saltveit, 1997).

2.6.2 Wound-ethylene synthesis Ethylene production is stimulated when plant tissues are subjected to

stress, such as from wounding. This ethylene has been named wound

ethylene or ‘stress ethylene’ (McGlasson and Pratt, 1964; Yang and

Hoffman, 1984). The stimulation of wound ethylene production can

occur over a short time frame, often within 1 hour of wounding, with

peak rates usually within 6 to 12 hours (Abeles et al., 1992a; Yang

and Hoffman, 1984; Yang and Pratt, 1978). Wounding increases the

activity of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and

results in the increased production of ACC, which can be oxidised to

ethylene (Boller and Kende, 1980; Yu and Yang, 1980). Mattoo and

Anderson (1984) and Abeles et al. (1992a) stated that membrane-

associated ACC may interact with membrane-bound ethylene-forming

enzymes (ACC oxidase). During this interaction, ACC is produced in

Slicing

Respiration

Reduced carbohydrates

& flavour changes

Ethylene

Ripening

Softening

Membrane deterioration

Loss of normal cell

function

Nutritional changes Water loss

Wound healing

Suberin

Pathological invasion

62

close proximity to the ACC oxidase that then uses the ACC to produce

ethylene.

As a consequence of cutting a tomato, large increases in ethylene

production have been observed by several researchers (Artes et al.,

1999; Brecht, 1995; Lee et al., 1970; Mencarelli et al., 1989).

Mencarelli et al. (1989) reported that slicing ‘breaker’ maturity tomato

fruit increased ethylene production 3-4 fold and increased the rate of

ripening compared with intact fruit. Slicing also caused increased

ethylene production in tomato disks (Lee et al., 1970). After 15 - 20

minutes of cutting the tomato into small disks, ethylene production

was approximately 20-fold higher than for the intact fruit (Lee et al.,

1970). Disks taken from different portions of the fruit have different

levels of ethylene production (Brecht, 1995). Within an individual

‘mature-green’ tomato fruit, tissue excised from the distal (blossom)

end entered the climacteric phase before the tissue taken from the

equator or proximal (stem-end) regions. Excised tissue from all

regions produced much more ethylene than intact fruit during the

climacteric phase (Brecht, 1995). Artes et al. (1999) showed that

ethylene production by tomato slices was higher than for whole fruit

at 10 °C from the first hour after slicing and up to 7 days in storage.

From the above studies, it has been shown that the level of ethylene

from tomato slices increases in proportion to the amount of wounding.

Ethylene resulting from wounding induces ripening process and

advances the onset of climacteric ethylene production (Saltveit, 1997).

Elevated ethylene after slicing in other fruits has been reported.

Varoquaux et al. (1990) reported that ethylene production from

kiwifruit slices decreased for 2 hour at 20 °C, then 2 - 4 hours later,

increased sharply, peaking at 7 times the rate of intact fruit, and

decreasing slightly or remaining constant after about 10 hours.

Watada et al. (1990) also showed ethylene production rate 16-fold

higher in sliced kiwifruit than in intact fruit. The continual increase

63

in ethylene production resulting from the slicing process was probably

due to the stimulation by endogenous ethylene. It was concluded that

ethylene production rates were proportional to the injured surface

area, and hence to the intensity of the stress (Watada et al., 1990).

McGlasson and Pratt (1964) showed a ten-fold increase in ethylene

production by cantaloupe flesh tissue slices compared with intact

fruit. Slices from nearly mature fruits showed a climacteric pattern,

and respiratory increase could be induced in slices treated with

ethylene.

2.6.3 Elevated respiration As a consequence of wounding in plant tissue respiration rate is

stimulated (Kays, 1991), but the initiation of this response is delayed

compared to that found for wound-induced ethylene (Brecht, 1995).

Asahi (1978) proposed that the increase in respiration is due to

enhanced aerobic mitochondrial respiration. Toivonen and DeEll

(2002) explained that increases in respiration is partially due to

removal of physical barriers (i.e. periderm or cuticle) to gas exchange

in the tissues. Another explanation for the rise in respiration was

stated by Laties et al. (1972). He demonstrated that in cut potatoes,

the rise in respiration after cutting or wounding is at least partially a

result of α-oxidation of long-chain fatty acids.

Increased respiration results in physiological changes. Saltveit (1997)

stated that enhanced respiration, coupled with decreased gas

diffusion due to liquid on the surface, can elevate carbon dioxide and

deplete oxygen to levels that stimulate anaerobic respiration. For

instance, shredded carrots are more susceptible to developing

anaerobic metabolism than whole carrots (Rolle and Chism, 1987).

Elevated respiration also causes a rapid decrease in stored reserves

(Saltveit, 1997). Laties (1978) demonstrated that starch breakdown is

64

enhanced, and both the tricarboxylic acid cycle and electron transport

chain are activated.

Increases in respiration as a consequence of cutting may be quite

substantial. Varoquaux and Wiley (1994) reported that the

respiration of fresh slices is, in most cases, 3 to 7 times that of the

intact organ, e.g. 4 to 7 times for grated carrots (Varoquaux and Wiley,

1994) and double for sliced and peeled ripe kiwifruit compared to

whole fruit (Brecht, 1995). Sliced strawberries and pears consumed

O2 at higher rates than whole fruit throughout 7 days of storage at

2.5 °C, and after transfer to 20 °C for 1 day (Rosen and Kader, 1989).

Short shelf life of the product is associated with higher respiration

rates (Kader, 1987; Rolle and Chism, 1987). Therefore, Watkins

(2002) suggested that keeping respiration rates at minimal levels is

desirable for maintaining quality.

Fresh-cut products generally have higher respiration rates than

corresponding intact products (Watada et al., 1996). Slicing of

‘mature-green’ tomatoes results in increased respiration by up to 40%

when stored at 8 °C, compared to the intact product (Mencarelli et al.,

1989). Watada et al. (1996) reported that respiration rates of fresh-

cut fruit increased with temperature. In the 0 to 10 °C storage

temperature range, the Q10 for tomato slices (7.1) was higher than

that for the whole product (2.9). Moreover, the Q10 was lower in the

10 - 20 °C temperature range (3.5) than in the 0 - 10 °C range (7.1).

Artes et al. (1999) reported that the respiration rates of fresh-cut

tomatoes increased significantly after 2 days at 10 °C. Cantwell

(1997) stated that higher respiration rates indicate a more active

metabolism and usually a faster deterioration rate. Higher respiration

rate can also result in more rapid loss of acids, sugars and other

components that determine flavour quality (Cantwell, 1997).

65

2.6.4 Membrane deterioration

Tomato tissue destined for fresh consumption begins to irreversibly

deteriorate following slicing. The process of cutting tissue into slices

damages the cells and cell membranes at the cutting surfaces

(Saltveit, 1997). Figure 2.11 depicts the events of membrane

deterioration in plant tissue.

Figure 2.11 Mechanism for membrane deterioration in plant tissue due to senescence or postharvest stress (Marangoni et al., 1996)

Wounding of plant tissue during the preparation of fresh-cut products

may cause membrane lipid degradation (Deschene et al., 1991; Rolle

and Chism, 1987). The ethylene produced by membrane systems may

play a role in this process by increasing the permeability of

membranes and reducing phospholipid biosynthesis (Watada et al.,

1990). Sheng et al. (2000) demonstrated in tomatoes that ethylene

production is a consequence of the metabolism of free fatty acids by

lipoxygenase, suggesting that the wound-induced membrane

66

breakdown may be directly associated with wound-induced ethylene

production.

Membrane deterioration results in plant tissue becoming vulnerable to

discolouration (Brecht, 1995; Watada and Qi, 1999b),

decompartmentation of cellular structure and organisation and loss of

normal cellular function (Brecht, 1995; Rolle and Chism, 1987). Rolle

and Chism (1987) stated that to maintain the quality of fresh-cut

products, membrane integrity must be maintained and the onset of

senescence must be delayed.

2.6.5 Wound healing Plant tissue can sometimes naturally seal the site of injury, such as in

potato and sweet potato (Brecht, 1995). The phrase wound healing is

used in some instances to refer to formation of suberin, callus, and

lignin production and deposition of cell walls at the wound site to form

a wound periderm (Burton, 1982; Kays, 1991). Suberisation of the

cell layers occurs in many tissues such as in cucumber pericarp and

in potato (Walter et al., 1990). To date, there is no information about

this process in tomato slices.

2.6.6 Water loss

Epidermis or skin is a very important barrier to water loss that would

otherwise lead to loss of turgor and desiccation. Fresh-cut fruit can

have large surfaces without any skin. In addition, processing leads to

an increase in the surface area/volume ratio. Therefore, fresh-cut

products tend to be more vulnerable to water loss (Brecht, 1995;

Garcia and Barrett, 2002).

67

Burton (1982) reported the differences in rate of water loss between

intact and wounded plant surfaces as varying 5 - 10-fold for organs

with lightly suberised surfaces such as carrots and parsnips, to

10 - 100-fold for organs with cuticularised surfaces such as spinach

leaf, bean pods, and cucumber fruit, and to as much as 500-fold for

heavily suberised potato tubers (Burton, 1982). Reduction of water

loss can be achieved by appropriate handling techniques including

lowering temperature and/or increasing the vapour pressure of the

surrounding air using packaging (Garcia and Barrett, 2002).

2.6.7 Susceptibility to micro-organisms Fresh-cut products provide an ideal media for the growth of micro-

organisms, so sanitation is essential to keep the microbial population

to a minimum (Watada and Qi, 1999a). Bolin et al. (1977) showed

that storage life is shorter with higher initial microbial loads. The

primary pathogen microorganisms living on fresh-cut products are

mesophilic microflora, lactic acid bacteria, fecal coliforms, yeasts and

molds, and pectinolytic microflora (Nguyen-the and Carline, 1994).

The sources of contamination in these products include the raw

material, the plant workers, the slicer, and the processing room

(Heard, 2002; Verlinden and Nicolai, 2000).

To reduce the contamination by microorganisms in fresh-cut

products, the whole and fresh-cut produce are generally washed by

50-200 µL L–1 chlorine solution (Watada and Qi, 1999a). After

pathogens have infected the products however, chlorination is not

very effective (Sawyer, 1978). If washing is conducted properly,

microbial populations can be lowered. In most studies on fresh-cut

tomato slices (Artes et al., 1999; Gil et al., 2002) the researchers used

sodium hypochlorite at 100 µL L–1. Although chlorination has no

residual effect (Sawyer, 1978), fresh produce exposed to pathogens

after treatment remains susceptible to re-infection (Hong and Gross,

68

1998). Another method to prevent microorganisms proliferation is low

temperature storage (Heard, 2002). However, storage of produce at

low temperatures does not eradicate microorganisms, it generally

reduces the growth rates of spoilage organisms and food-borne

pathogens (Heard, 2002; Verlinden and Nicolai, 2000).

Surface sterilisation of tomato fruit for sanitation purposes with

sodium hypochlorite has been found to alter firmness of tomato slices.

Hong and Gross (1998) showed that pericarp firmness of tomato slices

from fruit that had been treated with 1.05% sodium hypochlorite

(10500 µL L–1) for 60 seconds was less than one-half the firmness of

water-treated controls and lower than fruit pericarps from a 0.26 %

(2600 µL L–1) treatment. Artes et al. (1999) observed Rhizopus spp

colonies after 6 days storage of tomato slices at 10 °C. Hong et al.

(2000) observed the fungus Rhizopus stolonifer in slices from ‘red’

tomatoes grown using black polyethylene mulch with nofungicide

treatment after 12 days storage at 5 °C, and no fungi were detected in

tomatoes grown using black polyethylene with weekly fungicide

application. Blue mold rot, caused by Penicillium species, was

observed by Hong and Gross (2001) on fresh-cut tomato slices at the

end of 20 days storage at 5 °C. These results suggest that prevention

of microbial growth is an essential component in the storage of tomato

slices.

2.6.8 Loss of firmness In general, slicing fruit tissue results in loss of firmness (Varoquaux

and Wiley, 1994). The changes in texture of sliced fruits and

vegetables are either directly or indirectly affected by ethylene (Kader,

1985). Rosen and Kader (1989) have shown that wound-induced

ethylene production is associated with an increased rate of softening

in pear and strawberry slices.

69

The loss of firmness after slicing has been reported for apple slices

(Ponting et al., 1972), kiwifruit (Varoquaux et al., 1990) and kiwifruit

packed with banana sections (Watada et al., 1990). Varoquaux et al.

(1990) reported that kiwifruit slices lose 50% of their initial firmness

in less than 2 days at 2 °C. It was stated by Gross (1990) that

textural breakdown of kiwifruit slices during storage was due to

enzymatic hydrolysis of cell wall components. These results were

confirmed by Watada et al. (1990) who noted that the average firmness

of 1 cm-thick kiwifruit slices decreased by about 25% after 24 h and

by 40% after 48 h at 20 °C. They also reported that exposure of slices

to 2 or 20 µL L–1 ethylene accelerated the loss of firmness.

2.6.9 Flavour changes Flavour changes or flavour loss during and after processing are

especially of concern in fresh-cut products. Biochemical changes due

to wounding can affect flavour quality (John and Baldwin, 2002).

Fresh produce that exhibits high respiration due to wounding may

catabolize sugars or acids as a carbon source during storage (John

and Baldwin, 2002; Saltveit, 1997). According to Huxsoll et al. (1989),

flavour change may result from the loss of flavour compounds or from

the accumulation of compounds that produce off-flavours.

2.6.10 Nutritional changes Fresh-cut processing influences nutritional quality of produce (Klein,

1987). Mozafar (1994) and Lee and Kader (2000) maintained that if

vegetables are severely cut or shredded, as in the case of cabbage,

lettuce, carrots, and other vegetables sold as salad mixes, loss in

vitamin C or decrease in ascorbic acid content occurs. Cellular

disruption in fresh-cut vegetables and fruit increases enzymatic

activity by allowing substrate and enzymes to come in contact, and

results in rapid breakdown of vitamin C (Klein, 1987). Therefore,

70

Klein (1987) proposed that as long as cellular integrity remains,

vitamin content is not markedly changed. Mechanism of ascorbic acid

degradation is depicted in Figure 2.12.

Figure 2.12 Degradation of ascorbic acid (Klein, 1987)

The nutritional quality of fresh-cut products, including fresh-cut

tomato slices, changes as fruit maturity progresses. Kader et al.

(1977) reported that tomato fruit harvested green and ripened at 20 °C

to table-ripeness contained less ascorbic acid than those harvested at

the table-ripe stage. Betancourt et al. (1977) also demonstrated that

tomato fruit analyzed at the ‘breaker’ stage contained only 69% of its

potential ascorbic acid concentration if ripened on the vine compared

to table ripe-tomatoes. These results indicate the significant role of

maturity on the nutritional concentrations in products that are

subsequently processed. In kiwifruit, Agar et al. (1999) reported that

kiwifruit slices stored at 5 and 10 °C exhibited a gradual decrease in

ascorbic acid and an increase in L-dehydroascorbic acid. The total

71

vitamin C was 8%, 13%, or 21% lower than initial values in slices kept

for 6 days at 0 °C, 5 °C, or 10 °C, respectively.

Current information on lycopene bioavailability and stability of

lycopene in fresh-cut products is very limited. Barringer (2004)

suggested that the main cause of lycopene degradation is oxidation,

including oxidation during processing, but lycopene may also be

destroyed in processed tomato products by heating (Shi and Maguer,

2000). Fish and Davis (2003) evaluated the rate of deterioration of

lycopene in cut watermelon tissue during frozen storage. They found

that a small percentage of lycopene (4 - 6%), degraded during an

initial freeze-thaw, and then a loss of 30 - 40% lycopene occurred over

12 months storage at –20 °C and a loss of 5 - 10% over the same

period at –80 °C. Lycopene was slightly more stable in pureed

compared with diced watermelon tisue stored at –20 °C. Tavares and

Rodriguez-Amaya (1994) reported that the lycopene content in

concentrated tomato products is generally lower than expected,

because of losses during tomato processing.

2.6.11 Summary

Physiological changes in fresh-cut products are related to wounding of

tissue. The slicing of tomatoes therefore causes reactions that lead to

quality deterioration. It is obvious that slicing results in a significant

effect on physiological activities including flavour changes, softening,

nutritional changes, and pathological attack. Control of the wound

response is the key to providing a fresh-cut product with good quality

(Cantwell, 1997). It has been shown that low temperature storage is

an important component for reducing the wounding responses in

fresh-cut products. It is also evident from this review that research on

tomato slices needs to concentrate on the physiological and

biochemical changes during storage and how these affect the quality

72

of tomato slice products. There is therefore, a challenge to control all

of these deteriorative factors.

2.7 General Summary

2.7.1 Summary Fresh-cut products are new forms of processed fruit and vegetable

commodities. Tomato, because of its widespread utility, is an obvious

target for fresh-cut production. At the present time however, little

postharvest information is available about the physiological and

biochemical changes that may occur in fresh-cut tomato slices during

storage. The quality factors that are important for this product are

texture, flavour and nutritional value (Section 2.3).

The biochemical and physiological changes during ripening of intact

tomatoes and of tomato slices (Section 2.4) and the physiological

consequences of slicing tomatoes (Section 2.6) have been highlighted

in this review. Fresh-cut products may be subjected to a variety of

postharvest handling techniques to retain quality and extend storage-

life as described in Section 2.5. Deterioration of tomato slices during

storage could also be controlled by external modifications, including

physiological manipulation (harvest maturity) and environmental

manipulation (temperature) as well as by manipulation of ethylene

action using an ethylene inhibitor (1-MCP).

There are a number of key research areas that could potentially assist

in the development of fresh-cut tomato slices with high sensory

quality, long storage life, and satisfactory nutritional value. These key

research areas have been developed into the objectives of this thesis.

The major research objectives of this study will therefore focus on the

physiological (ethylene and respiration), biochemical (firmness, juice

73

colour, soluble solids, titratable acidity, and electrolyte leakage), and

nutritional (ascorbic acid and lycopene) changes that occur in fresh-

cut tomato slices during storage.

2.7.2 Objectives

The specific objectives of the research are:

1 To determine the effects of slicing on the postharvest physiology

of tomato slices (Chapter 4)

2 To study the quality changes in tomato slices taken from fruit at

different stages of maturity and stored at different storage

temperatures (Chapter 5)

3 To characterise the involvement of ethylene in the loss of slice

quality (Chapter 6)

4 To determine the efficacy of 1-MCP in maintaining quality of

tomato slices (Chapter 7)

5 To determine the effect of fruit maturity and 1-MCP on the

quality of tomato slices (Chapter 8)

6 To evaluate the effect of applying a brief heat shock to intact

tomatoes on the quality of slices (Chapter 9).

74

3. MATERIALS AND METHODS

3.1 Overview The general overview of the method during minimal processing and

assessments is depicted in Figure 3.1.

Figure 3.1 Generalised flow-chart for the preparation and measurements made

Store at controlled temperature

Selection of unblemished fruit, with uniform size, firmness and colour

Whole fruit washed with 100 ppm NaOCl solution for 1 min at 10 °C

Drained and kept overnight at 10 °C

Sliced (7 mm-thick) and drained at 10 °C

Allocate slices to treatments in ventilated plastic containers

Harvest tomato and transport within air-conditioned car

Firmness measurements

Homogenise for soluble solids, titratable acidity

and juice hue angle

Electrolyte leakage, ascorbic acid and

lycopene

Ethylene and respiration measurements

75

3.2 Tomato fruit

Tomatoes used were the cultivar ‘Revolution’, obtained from local

growers at Gatton supplied by Syngenta Seeds Pty. Ltd. This

indeterminate variety is a processing tomato newly selected for slicing.

It is multilocular and not juicy when sliced (Anonymous, 2002b).

Immediately after harvest, fruits were transported to the Postharvest

Laboratory, University of Queensland, Gatton, in an air-conditioned

car within 2 hours of harvest. Then fruit were carefully sorted for

absence of visual defects, as well as sorted based on the size and stage

of maturity. To minimise the diseases, whole fruit washed with 100

ppm NaOCl solution for 1 min, drained and kept overnight at 10 °C.

3.3 Assessments made before experimentation

Before starting each experiment, fruit fresh weight (g) was measured

using a balance (Sartorius, Germany), and fruit diameter (mm)

determined with a circumference meter. Firmness and colour were

evaluated in order to have uniformly mature material. Whole fruit

firmness was measured on each fruit using the Instron materials

tester (Autograph, Shimadzu AGS-H 500N) equipped with flat plates

and a probe. This equipment measured firmness based on the

resistance of the fruit to deformation. The maximum force required to

deform the fruit surface 5 mm by a 10-mm diameter spherical probe

with a head speed of 10 mm/min was determined, with the fruit lying

transversally and the plate positioned on the fruit equatorial zone

(Artes et al., 1999). Results were expressed in Newtons.

Surface colour of each fruit was measured using a Minolta CR-200

chromameter (Minolta Camera Co. Ltd. Osaka, Japan). To reduce

variability, six measurements of surface colour were made at the

equatorial, and the blossom-end and stem-end of the fruit.

76

3.4 Handling of slices Slices (7 mm thick) for all experiments were obtained after tomatoes

were sliced parallel to the equator region fruit with a commercial

slicing machine (Fasline®, model 919/927, Carol Stream Illinios, Plate

3.1). The tomato slicer and all equipment required for the handling

and preparation of tissue slices were sanitised before each experiment

by rinsing with 70% (v/v) ethanol.

Plate 3.1 The commercial slicing machine (Fasline®, model 919/927,

Carol Stream Illinios) used in experiments.

All slicing process and all operations associated with preparation and

handling of the tomato slices were conducted in a fresh-cut room at

10 °C to minimise contamination. In addition, in order to minimise

any contamination of the slices, sterile gloves were used in handling

tomato slices. After slicing, care was taken to prevent further damage

77

to slices as they were put into glass jars or into the plastic containers

for storage and quality assessments.

Each plastic container (high density polyethylene with length: 16.5

cm, width: 10.5 cm, depth: 6.5 cm) was capped with a lid perforated

with 2 holes (10 mm-diameters). The holes were packed with clean

cotton wool to assist in maintaining sterility, and to enable adequate

ventilation of the atmosphere inside (Wu and Abbott, 2002). Each

containers containing two layers of absorbent paper on the bottom to

prevent juice accumulation (Gil et al., 2002).

3.5 Experimental measurements

3.5.1 Ethylene and CO2 evolution

A static system using 1 litre glass jars was used to measure ethylene

and CO2 production (Kader, 2002a). Tomato slice samples were

weighed and the volume of the jars measured by water displacement.

The headspace was calculated as the difference between the volume of

the jar and the fresh weight of fruit. Background ethylene was

measured from an empty jar that was sealed along with the sample

jars. All the jars were flushed with fresh air for 5 minutes before lids

were closed and sealed. Ethylene and CO2 evolution was determined

from the headspace after incubation for 2 hours at storage

temperature or at treatment temperature. Headspace gas samples

were taken with a 1 mL disposable syringe through the gas sampling

port in the lids. Syringes were flushed at least 5 times with air

between sampling from different treatments. Then the syringes were

flushed 5 times with air from inside the glass jars to stir the internal

atmosphere before a sample was withdrawn. Syringes were regularly

disposed of to avoid potential contamination.

78

For ethylene determination, samples were injected into a gas

chromatograph (Shimadzu model GC-8A) fitted with a flame ionisation

detector. Temperatures of the injector port, column and detector were

120, 90, and 120 °C, respectively. The 900 mm-long and 5 mm

internal-diameter glass column was packed with activated alumina

mesh size 80/100. The Shimadzu CRGA Chromatopac integrator

output was calibrated using an ethylene standard gas (0.09 ± 0.02 µL

L–1, BOC Gases β-grade) and the balance gas was nitrogen. The

carrier gas (1 kg cm-2 pressure) was high purity nitrogen (BOC Gases).

Oxygen (0.3 kg cm-2) was supplied as medical grade air, and hydrogen

(0.45 kg cm-2) was high purity grade, both from BOC Gases.

Ethylene production rate on a fresh weight basis was calculated as

follows:

Ethylene production (nmol g -1 h-1) =

∆ C2H4 × wt(g) fresh

vol(L) space head×

t(h)1

× Ctemp273K

273K°+

× 22.41000

where:

∆ C2H4 = ethylene concentration in sample – background (µL L–1)

t = incubation time

For CO2 analyses, headspace samples were injected into a gas

chromatograph (Shimadzu model GC-8A) fitted with a thermal

conductivity detector. Temperatures of the injector port, column and

detector were 30, 35, and 30 °C, respectively. The 1.5 m-long and 1.8

mm-internal diameter glass column was packed with activated

alumina mesh size 80/100. The gas chromatograph signal was

recorded using a Shimadzu CRGA Chromatopac integrator calibrated

with a CO2 standard of 0.575% (v/v) in nitrogen (BOC Gases β-grade).

The carrier gas (1 kg cm-2 pressure) was high purity helium (BOC

Gases).

79

The rate of carbon dioxide production was used to indicate the

respiration rate. The respiration rate on a fresh weight basis was

calculated using the following equation:

Respiration rate (µmol g-1 h -1) =

∆ CO2 × 104 × wt(g) fresh

vol(L) space head × t(h)1 ×

Ctemp273K273K

°+ ×

22.41

where:

∆ CO2 = CO2 in sample – CO2 in background (% v/v).

t = incubation time

3.5.2 Slice firmness

Slices were brought from the storage room, then columella and

pericarp firmness was measured immediately before the sample

temperature could change. Columella firmness (Chapter 4 only) was

assessed at the centre of columella, whereas pericarp firmness was

assessed at the outer pericarp at two opposite locations using a

materials tester (Autograph, Shimadzu AGS-H 500N). The firmness

measurements were undertaken by placing each slice on a flat plate

held perpendicular to the probe. Firmness (N) was determined by

measuring the force required for a 4 mm-diameter cylindrical probe to

penetrate the cut surface 3 mm at a speed 1 mm/sec, following Wu

and Abbott (2002).

3.5.3 Soluble solids (SS), titratable acidity (TA) and ratio SS/TA

Juice was extracted from slices by homogenising them at high speed

for 1 minute in a food blender. The homogenate was filtered through

two layers of cheesecloth to obtain the clear filtrate. Soluble solids

content of the resulting clear juice (about 5 g) was determined at

80

20 °C using an Atago Digital Refractometer (PR-101, Fuji, Japan), in

units of °Brix. The refractometer was initially zeroed using distilled

water, and the prism was wiped with clean tissue paper and then

rinsed with distilled water after each measurement.

Titratable acidity was measured on 10 g of juice diluted with 50 ml of

distilled water. The diluted juice was stirred (IKAMAG, Janke &

Kunkel, Australia) then titrated with 0.1 N NaOH to an end point of

pH 8.2. An automatic titrator (Metrohm, Swiss) equipped with a 632

pH meter and 765 Dosimat Autoburette was used to measure

titratable acidity. Titratable acidity (TA) was expressed as percentage

(w/w) citric acid. The percent total titratable acidity as citric acid was

calculated by the following equation (Roberts et al. (2002) and Baker

(Queensland of Department of Primary Industry and Fisheries,

personal communication).

% citric acid = juice ofg 10

1000.064NaOH) of (normality 0.1(mL) vol.NaOH ×××

Where 0.064 = milliequivalent factor for citric acid

The ratio soluble solids to titratable acidity was calculated as

acidity titratable content solids soluble

3.5.4 Juice colour

Juice colour was measured using juice extracted as in section 3.5.3

using the CIELAB L*, a*, and b* values obtained with a Minolta CR-

200 (Minolta Camera Co. Ltd. Osaka, Japan) tristimulus colormeter.

Colour, as hue angle (hº), was measured by aiming the sensor through

the base of a glass jar containing 40 ml of juice (Artes et al., 1999).

81

Hue angle was calculated on the basis of the following equations

(Arias et al., 2000a; Lancaster et al., 1997):

h° = arctangent (b*/a*), when a* > 0 and b* ≥ 0

or

h° = 180° + arctangent (b*/a*), when a* < 0 and b* > 0;

3.5.5 Electrolyte leakage Electrolyte leakage rate was determined from conductivity

measurements according to the procedure of King and Ludford (1983)

and Hong et al. (2000). Four sections of pericarp discs (about 6 mm

diameter) were excised from each slice and combined. The discs were

weighed (about 2 g) and washed three times in distilled water and

placed in beakers containing 30 ml 0.4 M mannitol solution. In an

initial experiment it was found that conductivity of the bathing

solution did not increase appreciably with incubation for up to 6 h.

The discs were held at 30 °C for 6 hours and then 20 mL of solution

was taken and initial electrical conductivity readings were taken using

a conductance meter (Activon Model 301, Conductivity Meter,

Australia). The beakers were swirled for 10 s before the electrolyte

measurements were taken. The discs and bathing solutions were then

frozen at –20 °C for 24 hours and then thawed. Final conductivity

readings were taken and electrolyte leakage (%) was calculated

(McCollum and McDonald, 1991):

Electrolyte leakage (%) = 100 tyconductivi Finalty conductivi Initial

×

3.5.6 Ascorbic acid Ascorbic acid concentrations were determined using High Performance

Liquid Chromatography (HPLC, LC-10 AD Liquid Chromatograph,

Shimadzu, Japan) according to Rizzolo et al. (1984). Approximately 20

82

g of slices stored at -80°C were thawed and homogenised with a

homogenizer (Janke & Kunkel IKA, Ultra Tunax T25) at 13 500 rpm

for 2 minutes at room temperature. Ten gram of homogenate was

weighed and mixed with 25 ml of 6% (w/v) metaphosphoric acid. The

solution was centrifuged (P Selecta, Centronic, Spain) for 20 min at 6

000 rpm (2500 x g). The extract was transferred into a 100 ml

volumetric flask after filtration through Whatman No. 1 filter paper.

The residue from the filtration was extracted with metaphosphoric

acid once more and the second extract was combined with the first.

The mixture was filtered through Whatman No. 1 filter paper and the

filtrate was diluted to 100 ml with 6% (w/v) metaphosphoric acid. An

aliquot of the acid extract was then filtered through a Millipore filter

(Millex HA) prior to injection of 10 µL into the HPLC (SIL-10AXL,

Shimadzu, Japan). The ascorbic acid was separated on a column of

Luna 5µ C18 (Phenomenex, USA) with length 150 mm x diameter 4.6

mm, equipped with a guard column C18 5µ. The mobile phase was

0.2 N orthophosphoric acid, the flow rate was 0.8 mL/min, and the

detection wavelength was 254 nm. L (+) ascorbic acid (0.01 mg/mL)

(Merck) was used as the external standard for quantification. To

determine recovery of the procedure, a known amount of pure

ascorbic acid standard (i.e. 2 x the amount found normally) was added

to disc samples and then the extraction and chromatographic

procedures were applied to samples with and without standard

ascorbic acid, in duplicate. The recovery was 96 %, indicating

complete extraction. Ascorbic acid concentration on a fresh weight

basis was calculated:

Ascorbic acid (mg 100 g-1) =

sampleg g

standard of Area sample of Area 100×

83

3.5.7 Lycopene

Lycopene analyses were performed according to Beerh and Siddappa

(1959), Adsule and Dan (1979), and Hakim et al. (2000). Pigments

were measured by homogenizing ca. 3 g of frozen pericarp tissue with

a homogenizer (Janke & Kunkel IKA, Ultra Tunax, T25) at 13 500 rpm

in 10 ml of acetone in a centrifuge tube at room temperature. The

tubes used were covered with aluminium foil to prevent light-induced

lycopene oxidation. The tubes were shaken on a shaker (B.Braun,

Melsungen AG, Germany) for 15 min at 150 cycles/min and then

centrifuged (P Selecta, Centronic, Spain) at 6000 rpm (2500 x g) for 10

minutes at room temperature. The supernatant was decanted and

adjusted to 15 ml with acetone. Lycopene concentrations were

determined from the absorbance at 503 nm in an acetone extract

using a spectrophotometer (Pharmacia LKB, Ultrospec III, Japan).

This wavelength is best suited for tomato lycopene because the

influence of carotenoids is negligible (Beerh and Siddappa, 1959).

Lycopene content was calculated using the molecular extinction

coefficient of 17.2 x 104 mol cm-1 (Beerh and Siddappa, 1959;

Mencarelli and Saltveit, 1988) and was expressed on a fresh weight

basis:

Lycopene (mg kg-1) =

mL10L 1

moleg

cmΜ1017.2A

34503 ××

××9.536

1g mg 103

×tissuekg

mL 10.0×

tissuekg 0.0312 A ×

= 503

tissueg 31.2A 503 ×=

84

3.6 Statistics and data analyses Treatment means and standard errors of means were calculated using

Microsoft® Excel (Version 2002 Microsoft Inc.). Figures were drawn

using Sigmaplot® (Version 8). Analyses of variance were performed

using Minitab version 13.2 (2002) for Windows using the General

Linear Model. All measurements had equal sample size (balanced

data) and the least significant difference (LSD) procedure at P = 0.05

was used to test for differences between treatment means. Only

significant differences are discussed, unless otherwise stated.

Residual analyses of the data were performed to check that data

satisfied the assumptions of ANOVA. Analyses are presented in full in

the Appendix.