Studies on Storage Behaviour of Tomatoes Coated with

Studies on Storage Behaviour of Tomatoes Coated with

Chitosan-Lysozyme Films

Padmini Thumula

Department of Bioresource Engineering

Faculty of Agricultural and Environmental Sciences

McGill University

Montreal, Quebec, Canada

Jun. 02, 2006

A thesis submitted to the McGill University in partial fulfillment of the

requirements for the degree of Master of Science

©Padmini Thumula 2006 All rights reserved.

ii

ACKNOWLEDGMENTS

It is indeed a privilege to work under the leadership of my

supervisor Professor Dr. G.S.V. Raghavan. I deeply appreciate his

encouragement, guidance and patience that has helped me complete this

study. I am specially grateful to him for giving me this opportunity. My

sincere thanks to Dr. M. Ngadi, Professor for his interest and support.

I am indebted to Dr. Yvan Gariépy for all the technical assistance

and guidance in lab work. His sense of direction and scrupulous planning

and organization of work is admirable. My special thanks to Dr. Valérie

Orsat and Dr. Venkatesh Sosle for their encouragement and suggestions

throughout my study.

I would like to express my deep sense of gratitude to my friend Mr.

R. S. Satyanarayandev for his constant moral support and all the help

rendered during my study. To my family I owe much for their sustained

support and strength given to me.

I acknowledge the financial support rendered by the Canadian

International Development Agency.

iii

FORMAT OF THESIS

This thesis is submitted in the form of original papers suitable for

journal publication. The thesis format has been approved by the Faculty of

Graduate Studies and Research, McGill University, and follows the

conditions outlined in the "Guidelines Concerning Thesis Preparation,

section 7, Manuscripts and Authorship" which are as follows:

"The candidate has the option, subject to the approval of the Department, of including as part of the thesis the text, or duplicated published text (see below), or original paper, or papers. In this case the thesis must still conform to all other requirements explained in Guidelines Concerning Thesis Preparation. Additional material (procedural and design data as well as descriptions of equipment) must be provided in sufficient detail (e.g. in appendices) to allow a clear and precise judgment to be made of the importance and originality of the research reported. The thesis should be more than a mere collection of manuscripts published or to be published. It must include a general abstract, a full introduction and literature review and a final overall conclusion. Connecting texts which provide logical bridges between different manuscripts are usually desirable in the interests of cohesion.

It is acceptable for the thesis to include as chapters authentic copies of papers already published, provided these are duplicated clearly on regulation thesis stationary and bound as an integral part of the thesis. Photographs or other materials which do not duplicate well must be included in their original form. In such instances, connecting texts are mandatory and supplementary explanation material is almost always necessary.

The inclusion of manuscripts co-authored by the candidate and others is acceptable but the candidate is required to make an explicit statement on who contributed to such work and to what extent, and supervisors must attest to the accuracy of the claims, e.g. before the Oral Committee. Since the task of the Examiners is made more difficult in these cases, it is in the candidate's interest to make the responsibilities of authors perfectly clear. Candidates following this option must inform the Department before it submits the thesis for review."

iv

Contribution of the authors:

The authorship for both the papers in this thesis is T. Padmini,

G.S.V. Raghavan and Y. Gariépy. This study was performed by the

candidate and supervised by Dr. G. S. V. Raghavan of the Department of

Bioresource Engineering, Macdonald Campus, McGill University,

Montreal. The entire research work was done at the Postharvest

Technology laboratory, Department of Bioresource Engineering, McGill

University. Dr. Y. Gariépy was technically involved in the instrumentation

and control of all the experiments in this study, giving expert guidance in

the usage of equipment.

v

TABLE OF CONTENTS

ACKNOWLEDGMENTS………………………………………………………………………. ii FORMAT OF THESIS...………………………………………………………………………….iii TABLE OF CONTENTS...................................................................................................... v LIST OF TABLES...............................................................................................................vii LIST OF FIGURES............................................................................................................viii ABSTRACT……………………………………………………………………………………….ix

Chapter I Introduction........................................................................................................ 1 1.1 Hypothesis ................................................................................................................ 4 1.2 General Objectives ................................................................................................... 5

Chapter II Review of Literature ........................................................................................ 6 2.1 Postharvest changes in fruits and vegetables.......................................................... 6

2.1.1 Physiological changes....................................................................................... 6 2.1.2 Compositional Changes .................................................................................... 8

2.2 Post harvest treatments............................................................................................ 9 2.3 Tomato composition and post harvest treatment ................................................... 10 2.4 Extending shelf life of fruits and vegetables ........................................................... 16

2.4.1 Modified atmospheric storage ......................................................................... 18 2.4.2 Edible coatings ................................................................................................ 22

2.4.2.1 Film composition ...................................................................................... 23 2.4.2.2 Chitosan as edible film............................................................................. 27 2.4.2.3 Lysozyme as biopreservative................................................................... 42

Chapter III Respiration and Quality Changes in Tomatoes Coated with Chitosan – Lysozyme Films during Ambient Storage Conditions............................................... 47

3.1 Introduction ............................................................................................................. 47 3.2 Objectives ............................................................................................................... 50 3.3 Materials and methods ........................................................................................... 50 3.4 Results and Discussion .......................................................................................... 55 3.5 Conclusion .............................................................................................................. 75 3.6 References ............................................................................................................. 75

Chapter IV Effect of Chitosan-Lysozyme Coatings on the Quality of Tomatoes Stored at Low Temperature and High Humidity ........................................................ 82

4.1 Introduction ............................................................................................................. 82 4.2 Objectives ............................................................................................................... 85 4.3 Materials and methods ........................................................................................... 85

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4.4 Results and Discussion .......................................................................................... 89 4.5 Conclusion ............................................................................................................ 105 4.6 References ........................................................................................................... 105

Chapter V General Discussion and Conclusion.............................................................. 111 LIST OF REFERENCES................................................................................................. 113

vii

LIST OF TABLES

Tab. 2.1: Commercially available modified atmosphere packaging systems ...................19 Tab. 2.2: Properties and characteristics of polysaccharide barriers.................................24 Tab. 2.3: Food applications of Chitin, Chitosan and their derivatives...............................30 Tab. 2.4: Edible coating applications and functions..........................................................35 Tab. 2.5: Properties of chitosan (ch) – lysozyme (ly) films ...............................................46 Tab. 3.1: Mean L* values of tomatoes during storage at ambient conditions...................62 Tab. 3.2: Mean hue values of tomatoes during storage at ambient conditions ................63 Tab. 3.3: Mean final values (in N) for firmness of tomatoes during storage at ambient

conditions..........................................................................................................69 Tab. 4.1: Mean L* values of coated and uncoated tomatoes during storage ...................91 Tab. 4.2: Mean hue angles of coated and uncoated tomatoes during storage ................92 Tab. 4.3: Mean firmness (in N) of coated and uncoated tomatoes...................................98

viii

LIST OF FIGURES

Fig. 2.1: Structure of chitin ................................................................................................28 Fig. 2.2: Structure of chitosan ...........................................................................................28 Fig. 3.1: CO2 production of tomatoes stored at ambient conditions..................................55 Fig. 3.2: Respiratory quotient of tomatoes stored at ambient conditions..........................56 Fig. 3.3: Internal CO2 of tomatoes stored at ambient conditions .....................................58 Fig. 3.4: Internal O2 of tomatoes stored at ambient conditions.........................................58 Fig. 3.5: Changes in mean mass loss of tomatoes during storage...................................60 Fig. 3.6 : Mean scores for Appearance of tomatoes during

storage at ambient conditions...........................................................................65 Fig. 3.7: Mean scores for Defects in tomatoes during storage at ambient conditions......66 Fig. 3.8: Mean scores for Shrinkage of tomatoes during storage at ambient conditions..68 Fig. 3.9: Mean pH of tomatoes during storage at ambient conditions ..............................70 Fig. 3.10 : Mean Titratable Acidity of tomatoes during storage at ambient conditions .....71 Fig. 3.11: Mean Total Soluble Solids (%) of tomatoes during storage at ambient

conditions..........................................................................................................72 Fig. 4.1: Mean mass loss% of coated and uncoated tomatoes during storage...............90 Fig. 4.2: Mean score for appearance of coated and uncoated tomatoes .........................95 Fig. 4.3: Mean score for disease and defects in coated and uncoated tomatoes during

storage ..............................................................................................................96 Fig. 4.4: Mean pH of coated and uncoated tomatoes during storage............................100 Fig. 4.5: Mean titratable acidity of coated and uncoated tomatoes during storage ........101 Fig. 4.6: Mean total soluble solids of coated and uncoated tomatoes during storage....102

ix

ABSTRACT

Simple technologies are required for reducing the post harvest

losses of horticultural produce. Edible films are being studied extensively

for application on fresh and cut fruits and vegetables. Tomato, being a

very nutritious and important food and a highly perishable climacteric fruit,

this study was planned to investigate the application of chitosan films.

Chitosan is a biodegradable waste product from sea food and is safe for

consumption. With a view to broaden its antimicrobial activity it was

combined with lysozyme, a lytic enzyme. Since the edible films are

sensitive to changes in temperature and humidity, they were studied under

ambient and optimal conditions of storage.

This study showed that 1% chitosan was more suitable for

tomatoes for storage at both conditions of ambient and low temperature.

Respiration study showed that 1% chitosan treatments resulted in more

favorable levels of CO2 production and internal O2. This was reflected in

the quality of tomatoes held under these conditions. Two per cent chitosan

films were unsuitable due to their high CO2 production and low internal O2

levels. Spoilage was more apparent in this treatment. Lysozyme addition

did not show any additional benefit.

x

The research in this study has demonstrated that the selection of

edible films for horticultural produce needs to be integrated with the

requirement of storage conditions of the produce.

xi

ABRÉGÉ

Il est nécessaire de développer de simples pratiques visant à

réduire les pertes post-récoltes des produits horticoles. Des pellicules

alimentaires sont en développement pour les fruits et légumes frais ou

préparés. La tomate est un aliment nutritif hautement périssable qui

bénéficierait d’une telle pellicule protectrice. La présente recherche s’est

donc penchée sur la question en étudiant l’utilisation de films de

chitosane-lysozyme sur la tomate. Le chitosane est un composé propre à

la consommation humaine et biodégradable que l’on peut extraire des

résidus de production de l’industrie des produits de la mer. Afin d’élargir

son utilité, le chitosane a été jumelé à du lysozyme, une enzyme capable

de détruire la paroi cellulaire de nombreuses bactéries. Puisque ces films

comestibles sont sensibles à la température et à l’humidité, ils ont fait

l’objet d’une étude dans des conditions ambiantes et des conditions

optimales d’entreposage.

L’étude a démontré qu’un film composé de 1% de chitosane était le

plus favorable pour des tomates et ce pour les deux conditions

d’entreposage. L’étude de la respiration des tomates a démontré qu’un

film composé de 1% de chitosane était le plus favorable au contrôle des

niveaux de O2 et de production de CO2 tel que démontré par la qualité des

xii

tomates dans ces conditions. Les films composés de 2% de chitosane

étaient inadéquats avec une forte production de CO2 et de trop faibles

niveaux de O2, entraînant une baisse de la qualité des tomates et une

augmentation des pertes. La présence de lysozyme n’a pas été

concluante.

Cette recherche a donc démontré que la sélection de films

comestibles pour l’entreposage de produits horticoles doit être effectuée

dans une approche globale prenant en compte toutes les conditions lors

de la manutention.

1

Chapter I

Introduction

Huge post-harvest losses of fruits and vegetables are a matter of

grave concern for any country whose economy is agriculture based. But

this is a general phenomenon happening in almost every developing

country. Fruits and vegetables are highly perishable commodities that

require to be handled with much care to minimize losses. Because of their

high moisture content horticultural crops are inherently more liable to

deteriorate especially under tropical conditions. They are biologically

active and carry out transpiration, respiration, ripening and other

biochemical activities, which result in quality deterioration.

Losses during post harvest operations due to improper storage and

handling are enormous and can range from 20-50 percent in developing

countries (Kader, 1992). During peak seasons when horticultural crops

arrive in plenty at the market, prices slump bringing the farmer less profit.

These stocks are carelessly handled due to lack of appropriate storage

and transport facility. There should be enough processing industries to

utilize the surplus. Moreover the varieties available need to be suitable for

processing. Here agriculture may be characterized as disjointed.

Production is not linked with marketing. With perishable crops like fruits

2

and vegetables, storage, packaging, transport and handling technologies

are practically non-existent in most developing countries. Hence

considerable amount of produce is wasted. Every crop is worthy of its

investment only when it is utilized completely without losses.

The quality of the harvested fruits and vegetables depends on the

condition of growth as well as physiological and biochemical changes they

undergo after harvest. Fruits and vegetable cells are still alive after

harvest and continue their physiological activity. The post harvest quality

and storage life of fruits appear to be controlled by the maturity. If the fruits

are harvested at right maturity their quality is excellent.

Respiration plays a very significant role in the post harvest life of

the fruits. In most fruits the rate of respiration increases rapidly with

ripening as in climacteric fruits when senescence and deterioration of the

fruits begin. To extend the post harvest life of the fruits and delay ripening,

its respiration rate should be reduced as far as possible. Ethylene,

produced by some fruits as they ripen, promotes additional ripening of

produce. Thus an understanding of the factors which influence the rate of

respiration and ripening is indispensable to developing appropriate post

harvest technologies.

3

Besides, sanitation is important to protect produce against post

harvest diseases and the consumers from food borne illnesses. Every

horticultural crop has optimal storage temperature, humidity and modified

atmospheric conditions. The basic requirements for conditions during

transportation are proper control of temperature and humidity and

adequate ventilation. In addition, the produce should be immobilized by

proper packaging and stacking, to avoid excessive movement or vibration.

Packaging of fresh fruits and vegetables has a great significance in

reducing wastage. It provides protection from physical damage during

storage, transportation and marketing. However, over-use of non-

biodegradable plastic trays and wrapping materials, as often seen in

modern supermarkets, creates an extra burden of waste disposal and

damages the environment.

Recently edible films have been developed to extend the shelf

life of fruits and vegetables. This environment friendly technology wraps

the film closely around the fruit preventing respiration and transpiration,

thus slowing down senescence. Studies have shown that these films can

be incorporated with nutrients or preservatives and are functional in

various ways. With demand for more natural foods, bio-preservatives are

being added to the films making it more wholesome for the consumer.

4

Tomato is an important vegetable crop that is available throughout

the year in most of the tropical countries with seasonal peaks during

June–October. During the peak season they are available in plenty

resulting in distress sales; as prices come down and with few cold storage

facilities available much of it is wasted. It would be worthwhile to develop

a simple method that is feasible on a small farm for extending the shelf life

of this crop so that they can be stored and sold at a better price. Hence

this study aims at investigating the effect of an edible film like chitosan-

lysozyme (developed by Park et al., 2004) on the shelf life and

marketability of fresh tomatoes stored under different conditions of

storage. It is expected to bring about a better understanding of the

behavior of the films when exposed to different conditions of storage

namely, temperature and humidity.

1.1 Hypothesis

Chitosan and lysozyme bio-films help to extend shelf life of

tomatoes by reducing respiration. The performance of the films will be

influenced by the concentration of chitosan and lysozyme. Suitability of the

film will also depend on the conditions of storage, namely the temperature

and humidity.

5

1.2 General Objectives

The major objectives of the study are:

1. To assess the respiration and quality changes in tomatoes coated with

chitosan – lysozyme films during ambient storage conditions

2. To study the effect of chitosan-lysozyme coatings on the shelf life of

tomatoes stored at optimal conditions

6

Chapter II

Review of Literature

This study is about edible films and its effect on the quality of a

horticultural produce. This review will cover topics on postharvest changes

in general and tomatoes in particular, of edible films and specifically

chitosan and lysozyme films.

2.1 Postharvest changes in fruits and vegetables

2.1.1 Physiological changes

When a fresh horticultural produce is harvested, the processes of

life continue in a modified form. The crop can no longer replace food

materials or water, so it must draw on its stored reserves. When they are

used up, the fruit or vegetable undergoes an ageing process leading to

breakdown and decay. It will eventually become unacceptable as food

because of this natural rot. The principal normal physiological processes

leading to ageing are respiration and transpiration (FAO, 1989).

Respiration uses stored starch or sugar as long as they last.

Oxygen from the air breaks down carbohydrates into carbon dioxide and

water. This reaction produces energy in the form of heat. When the air

supply is restricted and the amount of available oxygen in the environment

falls to about 2 percent or less, fermentation instead of respiration occurs.

7

Fermentation breaks down sugars to alcohol and carbon dioxide, and the

alcohol produced causes unpleasant flavors in produce and promotes

premature ageing. Poor ventilation of produce leads also to the

accumulation of carbon dioxide around the produce. When the

concentration of this gas rises between one and five percent in the

atmosphere, it will quickly spoil the produce by causing bad flavors,

internal breakdown, failure of fruit to ripen and other abnormal

physiological conditions. Therefore, proper ventilation of produce is

essential. Ripening occurs when the fruit is said to be mature. It is

followed by ageing (often called senescence) and breakdown of the fruit.

There are fruits which show a rapid rise and fall in respiration rate

during ripening and are said to be climacteric, for example tomato and

mango. The non-climacteric fruits like pineapple, lime and grape do not

show such sharp rise and fall in respiration rate. Ethylene gas is produced

in most plant tissues and is an important factor in initiating the ripening

process (Irtwange, 2006).

Most fresh fruits and vegetables contain from 65 to 95 percent

water when harvested. Fresh produce continues to lose water after

harvest and it cannot be replaced. This causes shrinkage and loss of

weight. This high humidity level prevents moisture loss that may occur due

8

to increased respiration and lowered transpiration. When the harvested

produce loses 5 or 10 percent of its fresh weight, it begins to wilt and soon

becomes unusable. The rate at which water is lost from plant depends on

the difference between the water vapor pressure of the plant and the

pressure of water vapor in the air. To keep water loss from fresh produce

as low as possible, it must be kept in a moist atmosphere. Air flow helps to

remove heat of respiration but must be controlled to prevent moisture loss

(Elazar, 2004). Altering the relative humidity (RH) of the storage

environment may also delay senescence. Perishable fruit and vegetable

products should be maintained at RH levels of 90-95%.

2.1.2 Compositional Changes

Many changes in the composition of the fruit may occur during

development, maturation and ripening on the plant. Some may continue

after, or start only at harvest. These changes being either desirable or

undesirable, can take place as loss of chlorophyll and development of

other colored pigments like carotenoids (yellow orange and red colors),

anthocyanins and other phenolic compounds. Starch is converted to

sugars which increase in fruits during ripening. In most commodities

starch is used as a substrate for respiration. Acidity decreases with

ripening and senescence. Acids and sugars are important for development

9

of fruit flavor. During ripening, softening occurs and polysaccharides such

as pectins, cellulose and hemicellulose are degraded by enzymes. There

are changes in proteins, amino acids and lipids which may affect the flavor

of the commodity. Development of flavor and aroma volatiles is very

important for acceptable eating quality. Loss of vitamins, especially

ascorbic acid (vitamin C), takes place during storage and thus adversely

affects nutritional quality (Irtwange, 2006).

2.2 Post harvest treatments

Fruits, vegetables and cut flowers are living and continue to respire

after separation from the parent plant. Their post-harvest life depends on

the rate at which they use up their stored food reserves and their rate of

water loss. When food and water reserves are exhausted, the produce

dies and decays. Holding them at their lowest safe temperature (0℃ or 32

°F for temperate crops or 10-12 ℃ or 50-54°F for chilling sensitive crops)

and relative humidity (60-90%) will enhance storage life by lowering

respiration rate, decreasing sensitivity to ethylene and reducing water

loss. Water loss results in shriveling and wilting, causing severe post

harvest losses (Krochta and Mulder-Johnston, 1997).

Chilling injury should be avoided, as they may fail to ripen (bananas

and tomatoes), develop pits or sunken areas (oranges, melons and

10

cucumbers), show brown discoloration (avocados, cherimoyas, eggplant),

increase susceptibility to decay (cucumbers and beans), and develop off-

flavors (tomatoes). In general proper storage practices include

temperature control, relative humidity control, air circulation and

maintenance of space between containers for adequate ventilation, and

avoiding incompatible product mixes (Shewfelt, 1990).

Sanitation is another essential factor, both to control pathogen

contamination and food spoilage. Chlorine treatments (100 to 150 ppm Cl)

can be used in wash water to help control pathogen buildup during

packing operations. A rule of thumb is to use 1 to 2 ml of chlorine bleach

per liter (1 to 2 ounces of chlorine bleach per 8 gallons of clean water).

Walls, floors and packing equipment can also be cleaned using

quarternary ammonium compounds labeled as safe for food processing

equipment (Kupferman, 1990).

2.3 Tomato composition and post harvest treatment

Tomato (Lycopersicum esculentum) is a warm-season crop. In

2005, Mexico, United States and Turkey were the world’s leading

producers of tomatoes according to the FAOSTAT database. World

tomato production was 125 Mt in 2005, of which 83 Mt came from

11

developing countries. India produced about 7.6 Mt in 2005

(http://faostat.fao.org).

Tomatoes were ranked highest in a comparison of crops and their

contribution of nutrients to the diet (Wills 1981). Tomatoes also provide

potassium, iron, phosphorus, and some B vitamins, and are a good source

of dietary fiber. Ripe tomatoes are red in color because they contain

lycopene, an antioxidant. Lycopene is a pigment synthesized by plants

and microorganisms. It has twice the ability as that of beta-carotene and

10 times that of alpha-tocopherol to quench singlet oxygen, which is a

metastable state of molecular oxygen (O2) responsible for oxidative

reactions (Rao and Agarwal 1999).

Water comprises 90% of the fresh weight of tomato fruit; and the

size of the fruit is influenced by the availability of water to the plant. The

large amount of water also makes the fruit perishable. As the tomato fruit

develops, starch decreases while carbohydrates such as sucrose and

reducing sugars increase (Jones 1999). Sugars are mostly found in ripe

fruit; and starches are found mostly in unripe fruit (Wills 1981). In a ripe

tomato, solids form about 5-7% of the fruit. About half of the solids

comprise sugars and one eighth is acids. The main sugar in tomatoes is

glucose. Citric acid is the main acid in tomato juice; and the pH of fruit is

12

normally between 4.0 and 4.5. The pH of the fruit increases throughout

development.

Vegetables or fruits with natural coatings of wax have lower

respiration rates than fruits without such protective barriers. Transpiration

is the movement of water through the cellular tissue of a plant, and

eventual evaporation of this water from plant surfaces. This movement of

water is driven by the gradient existing between the tissue of the plant and

the humidity of the surrounding air (Ben-Yehoshua, 1987). Transpiration

serves two purposes: first transpiration contributes to the lowering of the

surface temperature of the plant's tissues by evaporation. The second

function of transpiration relevant to post harvest is the maintenance of

turgidity of the plant's tissues and fruits. As much as 90% of the water

moving into a plant can be lost through transpiration. Plants have

therefore developed specialized tissue structures for preventing moisture

loss. When a fruit is removed from the plant, the replenishing water

source, the soil, is cut off and turgor is altered. The speed at which

damage from loss of turgidity occurs depends on the characteristics of the

commodity, including its rate of respiration, size, and state of maturity.

Respiration produces water and heat, both of which directly affect

transpiration. The metabolic water produced through respiration remains

13

within the fruits' tissue; however, the carbon dioxide lost to the air through

open stomata can result in weight loss of harvested fruits. Heat generated

during increased respiration after harvest may also contribute to weight

loss of a fruit. The heat lost to the environment contributes to increased

evaporation of water.

Water losses from transpiration may also be affected by the stage

of fruit maturity. In general, climacteric fruits have increased transpiration

at very early (pre-climacteric) stages. Increased transpiration also occurs

at the beginning of the climacteric phase.

Fruits and vegetables have colored pigments. The green colored

chlorophyll pigments are contained within the chloroplast. This pigment

may also be lost through photo degradation, which occurs when

chlorophyll molecules are bleached by light and oxygen. This process

occurs during ripening and senescence. Carotenoids are pigments with

colors ranging from yellow to orange red. The important pigments in

tomatoes are the lycopene and the beta carotene (Jones 1999).

Texture is imparted by components of plant tissue and its cell walls.

The cellular walls are made up of cellulose fibers which are held together

by cement like substance called pectin. These cells take up water, which

generates a hydrostatic pressure, giving rise to the crisp texture of

14

vegetable and fruit products. After harvest several factors affect the

texture of fruit and vegetable products. First, turgor pressure, and hence

crisp texture is altered. Turgor pressure change results from decreased

transpiration and respiration. Because additional water can not move into

the plant cells, and water still is being continually lost from the plant's

surface, wilting occurs. Softening of fruits and vegetables is brought about

by enzymatic dehydration of the pectin holding adjacent cells together. As

the fruit begins to senesce and proceed to an overripe stage, the pectin is

being changed into pectic acid by the enzyme pectinase. Pectic acid

imparts the characteristic mushy texture to overripe fruit (Whitaker, 1996).

Tomato is a short duration crop and it gives high yield; it is

important from economic point of view and hence area under its cultivation

is increasing day by day. It is not uncommon to hear that farmers in

developing countries like India dump cartloads of tomato on the streets.

Excess production results in a crash in tomato prices, with prices slumping

to 50 paise a kilo (one Canadian cent is equivalent to 42 paise), farmers

are left with no choice. Ever since the Agreement on Agriculture of the

World Trade Organization began to be debated in the country, increasing

agricultural productivity and improving food quality are being considered

as the only solution for farmers. There is good scope for increased

15

utilization of tomatoes but this can be obtained only by adopting suitable

storage techniques to avoid losses (www.indiatogether.org).

Simple, low cost technologies can be more appropriate for small

volume, limited resource commercial operations, for farmers involved in

direct marketing, for home gardeners, as well as for handlers in

developing countries. Local conditions for small-scale handlers may

include labor surpluses, lack of credit for investments in post harvest

technology, unreliable electric power supply, lack of transport, storage

facilities and/or packaging materials, as well as a host of other constraints.

Bruising, moisture loss, chilling injury, compositional changes, over

ripening, softening and decay are caused by harvesting at improper

maturity, rough handling, inadequate cooling and temperature

maintenance and lack of sorting. Utilizing improved post harvest practices

often results in reduced food losses, improved overall quality and food

safety, and higher profits for growers and marketers (Talukder et al., 2003;

Kitinoja and Kader, 1995)

Depending on the market and production area, tomatoes are

harvested at stages of maturity ranging from mature-green stage through

full-ripe. There are six stages of tomato fruit development during ripening

(for red fruited cultivars): mature green, breaker, turning, pink, light-red,

16

and red. Fruit change from green to red, due to the conversion of

chloroplasts, which contain chlorophyll to chromoplasts, which have red or

yellow carotenoids. Greenhouse-grown tomatoes are generally harvested

at various stages of maturity after mature green stage. Losses can occur

by improper packing through dropping, compression, vibration and

puncture (Walker1992). The fruits are sorted, graded and packed in lidded

cartons or plastic crates that can be stacked in a cold room for precooling.

Tomatoes are routinely palletized and cooled to 20 °C (68 °F) for ripening

or to 12 °C (53.6 °F) for storage. Optimal storage temperatures depend on

the maturity stage of the tomatoes. Optimal conditions for ripening are 19

to 21 °C (66 to 70 °F) with 90 to 95% RH (http://www.ba.ars.usda.gov).

According to Kader (1993) tomatoes are classed as highly perishable

crops. Mature-green tomatoes can be stored at18-220 C at 90-95% RH for

1-3 weeks and firm-ripe ones at 13-150 C at 90-95% RH for 4-7 days.

Chilling injury can occur at 7-100 C in ripe fruits and at 130 C in mature

green tomatoes, developing poor color and alternaria rot.

2.4 Extending shelf life of fruits and vegetables

The aim of applying postharvest technology to fruits and vegetables

is to maintain quality and to reduce losses between harvest and

consumption. Most often hi-tech practices are not suitable for small-scale

17

farmers in developing countries for the simple reason of economies of

scale (Kader, 1992). Limitations faced by small-scale handlers may

include labor surpluses, lack of credit for investments in postharvest

technology, unreliable electric power supply, lack of transport, storage

facilities and/or packaging materials and other constraints. There are

simple postharvest technologies that may meet the requirements of small-

scale food handlers. Despite good management practices, some produce

require treatment to prevent spoilage especially by pathogenic

microorganisms. This is done by hot water dipping, chilling, exclusion of

oxygen and use of chemical or bio pesticides (Shewfelt, 1986). The major

factors responsible for extending the shelf life of fruits and vegetables

include: careful harvesting so as not to injure the product, harvesting at

optimal horticultural maturity for intended use, and good sanitation

(Moleyar and Narasimham, 1994; Lee et al., 1996).

The delay in deterioration due to senescence is the main goal in the

preservation of fresh produce, as senescence accounts for the majority of

post-harvest losses (Lee et al., 1996). Control measures taken to minimize

production of ethylene following harvest include storage in a modified

atmosphere at optimal low temperatures (just above the chilling or

freezing injury threshold) and oxidizing the ethylene by various chemical

18

and physical means. Successful control of both product respiration and

ethylene production and perception by MAP can result in a fruit or

vegetable product of high organoleptic quality; however, control of these

processes is dependent on temperature. Controlled or modified

atmosphere storage should be used as a supplement to, and not as a

substitute for, proper temperature and relative humidity management.

2.4.1 Modified atmospheric storage

Ripening can be checked by using controlled atmosphere storage (CAS)

and/or active or passive modified atmosphere packaging (MAP) or with

edible coatings. A modified atmosphere can be defined as one that is

created by altering the normal composition of air (78% nitrogen, 21%

oxygen, 0.03% carbon dioxide and traces of noble gases) to provide an

optimum atmosphere for increasing the storage length and quality of

food/produce (Moleyar and Narasimham 1994; Phillips 1996). In all cases

an atmosphere of low oxygen (1-5%) and high carbon dioxide is created to

help reduce the respiration rate of fruits and vegetables and depress

ethylene production, thus preventing ripening (Lee et al., 1996). Reducing

the rate of respiration by limiting O2 prolongs the shelf life of fruits and

vegetables by delaying the oxidative breakdown of the complex substrates

which make up the product. However, at extremely low O2 levels (<1%),

19

anaerobic respiration can occur, resulting in tissue destruction and the

production of off-flavors and off-odors (Zagory, 1995), as well as the

potential for growth of food borne pathogens such as Clostridium

botulinum (Austin et al., 1998). Therefore, the recommended percentage

of O2 in a modified atmosphere for fruits and vegetables for both safety

and quality falls between 1 and 5%. CO2 can inhibit ethylene action as

well as autocatalytic production of ethylene by climacteric products such

as apples and tomatoes (Lee et al., 1996). The rate of respiration of a fruit

or vegetable is inversely proportional to the shelf life of the product; a

higher rate decreases shelf life.

Tab. 2.1: Commercially available modified atmosphere packaging systems

Product Description Use

Pallet Package System.

Pallet box wrapped in heavy gauge polyethylene, with a silicone membrane window to allow gas exchange regulation and a calibrated hole for pressure regulation

Apples, pears and other perishables

20


Marcellin System

For room storage: regulates the atmospheric composition via a parallel series of rectangular bags of silicone rubber; can be installed in or out of storage area and maintains a fairly consistent atmosphere.

Various perishables

Atmolysair System

System of gas diffusion panels enclosed in an airtight container, having two separate airflow paths and a control panel, allowing the potential for automation.

Cabbage in Canada, other perishables

Tectrol System (TransFRESH Co.)

Pallet box bulk unit-wrapped with a barrier plastic film; gases are injected and the bag-sealed.

Strawberries for short term transport

Tom-Ah-Toes (Natural Pak Produce)

Long, narrow box overwrapped with gas permeable film; contains a sachet containing calcium chloride and activated lime to absorb CO2.

Avocados, tomatoes, mangoes

MaptekFreshTM (SunBlush Technologies Inc.)

Maptek FreshTM is a post-harvest biotechnology where specific features and conditions are applied for each type of product to stabilize the produce and place it in a state of hibernation.

Fresh-cut produce: pineapple, fruit salad, cut tomatoes, mango, kiwi, melon, citrus fruits

21


MAPAX ® (AGA, Sweden)

This system incorporates the optimal atmosphere by testing, to choose the exact gas mixture and the best film for each product considering respiration rate, temperature, packaging film, pack volume, fill weight and light.

Fresh-cut produce, lettuce, mushrooms, pre-peeled potatoes

FreshHold (Hercules Chemical Co.)

Polypropylene label with calcium carbonate embedded in it.

Broccoli, asparagus, cauliflower and cherries

Cryovac (W.R. Grace and Co.)

0.75, 1.25, 2.5 mm thick bag made of several layers of polyethylene related polymers.

Cut lettuce, broccoli, cauliflower, spinach, peeled potatoes and other fresh fruits and vegetables

P-Plus films (Courtaulds Packaging)

Spark perforated films which result in non-uniform perforations throughout the film to facilita

te gas exchange.

Brussels sprouts, lettuce, broccoli, fresh mushrooms, and bean sprouts

(Church, 1993; Baldwin, 1994; Zagory, 1995; Lee et al., 1996; Raghavan et al., 1996; Smith and Ramaswamy, 1996; Padgett et al., 1998; Han, 2000 cited by Kitinoja and Kader, 1995)

22

A lot of research has gone into developing films for creating

suitable atmospheres for horticultural crops (Tab.2.1). Modified

atmospheric packaging of cauliflower, asparagus and carrots has been

studied and modeled using gas diffusion channels and silicone

membranes to regulate and maintain the gas composition at desirable

levels during storage (Reeleder et al.; 1989; Gariépy et al., 1991; Ratti et

al., 1996 and Ratti et al., 1998).

Ben-Yehoshua (1993) stated that perforated films enable MAP to

maintain water-saturation to prevent moisture loss, with only a slight

change in oxygen and carbon dioxide. ‘Smart’ films have been developed

to bring about the required atmosphere with in the package to suit the

produce. Edible biodegradable coatings are yet another modification of the

smart film technology, where a film is used as a coating directly on the

food (Guilbert et al., 1996). These films are gaining popularity due to both

environmental pollution and food safety concerns (Padgett et al., 1998).

2.4.2 Edible coatings

An edible film is defined as a thin layer of material which can be

eaten by the consumer, be applied on or within the food by wrapping,

dipping, brushing or spraying and act as barriers against transmission of

gases, vapors and solutes and provide mechanical protection (Wu et al.,

23

2002). Application of edible films to fresh produce has been one method of

extending its shelf life by slowing down its metabolic processes. This is

reported to have been in use since the 19th century (Kester and Fennema,

1986). With the advent of new materials for use as coatings and consumer

demand for more naturalness in food, environment friendly biofilms have

become popular and a very wide list of applications has been found.

Biobased packaging materials include both edible films and edible

coatings along with primary and secondary packaging materials

originating from agricultural and marine sources (Petersen et al, 1999;

Cha and Chinnan, 2004).

2.4.2.1 Film composition

Active packaging technology is a relatively novel concept designed

to provide interaction between food and packaging material, while

sustaining the microenvironment contained within. It is aimed at extending

the product shelf life, maintaining its nutritional and sensory quality, as

well as providing microbial safety. Many researchers have reviewed the

existing active products and patents (Vermeiren et al., 1999; Floros et al.,

1997; Alvarez, 2000). The main cause of spoilage of many foods is

microbial growth on the product surface. The application of antimicrobial

agents to packaging can create an environment inside the package that

24

may delay or even prevent the growth of microorganisms on the product

surface and, hence, lead to an extension of the shelf life and/or the

improved safety of the product.

Components of edible films and coatings can be divided into three

categories: hydrocolloids, lipids, and composites. Hydrocolloids include

proteins and polysaccharides, such as starch, alginate, cellulose

derivatives, chitosan, and agar. Lipids include waxes, acylglycerols, and

fatty acids. Composites contain both hydrocolloid components and lipids.

The choice of materials for a film or coating is largely dependent on its

desired function. Some of the edible films developed are shown in Tab.

2.2.

Tab. 2.2: Properties and characteristics of polysaccharide barriers

Film Film Preparation Advantages Disadvantages

Pectin- most effective on low moisture products

- generally made from low-methoxyl pectin, calcium chloride (cross-linker), a plasticizer, and sometimes organic acids

- can retard water loss from food

- can improve handling and appearance of foods

- not adequate moisture barriers

- low oxygen permeability

25


Chitosan – Nutri-Save (NovaChem) used for whole apples and pears

- methylation of the chitosan polymer results in increased resistance to CO2 permeability

- use of chitosan with lipids may solve moisture barrier problems

- natural preservative, inhibits growth of fungi

- impermeable to gases at 70% RH

- at 100% RH, permeability to CO2 and O2 due to diffusion with water

Derivatives of cellulose - Tal Pro-long (Courtaulds Group) - Semperfresh (Surface Systems Intl, Ltd.)

- used mainly for composite coatings comprised of the sodium salt of carboxymethyl cellulose (CMC) as the film former, with sucrose fatty acid ester as the emulsifiers

good - good film formers due to linear structure of polymer backbone - O2 - O2 is limited in entering the fruit more than CO2 is from escaping; limiting buildup of harmful CO2 and maintenance of reduced O2

- not good barriers to movement of water; however, the film can retain a moisture layer which will delay moisture loss from the fruit by being the first layer of moisture lost

26


Carrageenan Coatings - sucessfully used on cut grapefruit halves (Bryan 1972)

- extracted from several species of red seaweeds and used in food systems as a gel

- can reduce moisture loss, oxidation or disintegration of the product

- not yet approved by the FDA for food coatings

Baldwin et al., 1995

Plasticizers of food grade like glycerol, sorbitol, mannitol and

polyethylene glycol are added to the film forming solutions to increase its

flexibility. Solvent removal technique is commonly used for forming the

films. In this process a continuous structure is formed and stabilized by

physical and chemical interactions between molecules. The

macromolecules in the film forming solution are dissolved in a solvent

such as water, ethanol or acetic acid that contains the plasticizer. The film

forming solution is either cast into film and dried or is applied over the food

and then allowed to dry (Cagri et al., 2004).

The use of edible films and coatings to extend shelf life and

improve the quality of fresh, frozen and fabricated foods has been

extensively studied owing to its ecofriendly nature. They provide a means

of controlling the physiological, morphological and physicochemical

properties in foods to preserve them (Kittur et al., 1998). Polysaccharide

27

and protein film materials are characterized by high moisture permeability,

low oxygen and lipid permeability at lower relative humidities, and low

barrier and mechanical properties at high relative humidities (Brody,

2004).

Current research on edible films aims at facilitating formation of

films using new materials with improved properties. The films might be

formed into stand-alone packaging or coatings on foods. Even more than

thermoplastic materials, edible films may have the potential for

incorporation of further functional entities such as antimicrobials,

antioxidants, flavors, and nutrients (Brody, 2005).

2.4.2.2 Chitosan as edible film

Chitin, a cationic polysaccharide (Fig.2.1), (1, 4-linked 2-deoxy-2-

acetoamido-α-D-glucose) is a major component in the exoskeletons of

insects and the shells of crustaceans (crabs, shrimp and crayfishes),

making it easy to obtain. Chitosan (poly-beta-1, 4-glucosamine), the

deacetylated form (Fig. 2.2) of chitin can be obtained from crustacean

shells either by chemical or microbiological processes and is also

produced by some fungi (Aspergillus niger, Mucor rouxii, Penecillium

notatum) (Simpson, 1994).

28

Fig. 2.1: Structure of chitin

Fig. 2.2: Structure of chitosan

In general, chitosan has numerous uses: flocculant, clarifier,

thickener, gas-selective membrane, plant disease resistance promoter,

wound healing promoting agent and antimicrobial agent. Chitosan also

readily forms films and, in general, produces materials with very high gas

barrier, and it has been widely used for the production of edible coating

(Krochta and Mulder-Johnston, 1997). Furthermore, chitosan may be used

as coatings for other biobased polymers lacking gas barrier properties.

Chitosan is not digestible by the human body, and so ingesting it does not

increase calorie intake (Castro et al., 1996). Chitosan is well known to

have an antimicrobial activity against various microorganisms (Muzzarelli

and Muzzarelli, 2003). Even though it is still not used as a food additive,

29

its advantageous properties still attract the attention of food

manufacturers. Studies showed chitosan could be released from the film

matrix and inhibit microbial growth. Apart from its antimicrobial effect,

chitosan is also used in food as (1) clarifying agent in apple juice (2)

antioxidant in sausages (3) enzymatic browning inhibitor in apple and pear

juices and in potatoes and (4) antimicrobial agent in minimally processed

foods (Shahidi et al., 1999).

As it forms semipermeable films, chitosan can modify the internal

atmosphere (by altering the permeability to water, oxygen and carbon

dioxide), thereby decreasing the transpiration loss, reducing respiration

rate and delay ripening in fruits, maintaining the quality of harvested fruits

and reducing mold growth. Chitosan is good for fresh-cut fruits and

vegetables when it is in close contact with the tissue. Chitosan could be

an ideal preservative coating because of its film forming properties,

biochemical properties, inherent antifungal properties and elicitation of

phytoalexins (Kittur et al., 1998, El Ghaouth et al., 1992, Ghaouth et al.,

1991, Zhang and Quantick, 1997, Zhang and Quantick, 1998, Jiang and

Li, 2001, Li and Yu, 2000). Application of chitosan films in food is given in

Tab. 2.3.

30

Tab. 2.3: Food applications of Chitin, Chitosan and their derivatives

Area of application Examples

Antimicrobial agent Bacteriocidal, fungicidal

Edible film industry Controlled moisture transfer between food and surrounding environment, controlled release of antimicrobial substances, antioxidants, flavors and drugs.

Reduction of oxygen partial pressure, controlled rate of respiration, controlled enzymatic browning in fruits. Reverse osmosis membranes

Additive Clarification-deacidification of fruits and beverages.

Natural flavor extender, texture controlling agent, food mimetic, thickening and stabilizing agent, color stabilization.

Nutritional quality Dietary fibre.

Hypocholesterolemic effect

Livestock and fish feed additive

Production of single cell protein

Antigastric agent

Infant food ingredient

Recovery of solid materials from food

processing waste.

Affinity flocculation

Fractionation of agar

Water purification

Tharanathan R. N. and Kittur F. S., (2003)

31

Antimicrobial activity

Chitosan can be used as an antimicrobial film to cover fresh fruits

and vegetables. Chitosan activates several defense processes in the host

tissue, acting as a water-binding agent and inhibiting various enzymes

(Tharanathan and Kittur 2003). Generally, Gram-negative bacteria

seemed to be very sensitive to chitosan while the sensitivity of the Gram-

positive bacteria varied greatly. The antimicrobial activity is higher at low

pH. Muzzarelli (1990) states that the polytcationic nature of chitosan

allows interaction and formation of polyelectrolyte complexes with acid

polymers on the bacterial cell surface. In some fungi it can bring about

alterations in membrane functions, leading to changes in permeability,

metabolic disturbances and eventually death.

The antimicrobial activity increased with increasing concentration of

chitosan in the film matrix. Both gram positive and gram negative bacteria

were inhibited by those antimicrobial films when the incorporation of

chitosan was above 1.43%. The antimicrobial effectiveness of films tested

was decreased with increasing storage time. The controlled release of

chitosan from film matrix seems to effectively inhibit bacterial growth if the

amount of chitosan in film matrix is sufficient to inhibit the recovery of

microbial growth (Park et al., 2002). In general, the chitosan-based

32

antimicrobial films had no or little effect on the lactic acid bacteria, whether

inoculated or indigenous. Chen et al. (1996) and Ouattara et al. (2000)

reported that chitosan films made in dilute acetic acid solutions are able to

inhibit the growth of Rhodotorula rubra and Penicillium notatum by direct

application of the film on the colony-forming organism. Chitosan in organic

acids have displayed antagonistic effects against Escherichia coli,

Staphylococcus aureus, Saccharomyces cerevisiae, Enterobacteriaceae

and Serratia liquefaciens, whereas lactic acid bacteria were not affected.

Effective edible films formed as coatings on foods by dipping,

spraying, or panning could reduce packaging requirements and waste.

Chitosan films are clear, tough, and flexible and form good oxygen

barriers by casting from aqueous solution. Above all it is biodegradable

and environment friendly (Shahidi et al., 1999).

Physical properties of chitosan films

There are some basic factors to be considered before the

formulation of an edible coating. Two of these factors are the mechanical

structure of the film and the affinity between the coating material and the

fruit. Coating of fruits may be achieved by immersion, spraying, or

brushing followed by drying and cooling. When coating materials are

placed on the surface of fruits, two forces develop: cohesion of the

33

molecules within the coating, and adhesion between the coating and the

fruit. The degree of cohesion of the coating governs barrier and

mechanical properties of the coating. The higher the cohesion, the higher

the barrier properties and the lower the flexibility of the film (Guilbert and

Biquet, 1996). On the other hand, the degree of adhesion depends on the

chemical and electrostatic affinity of the coating material with the surface

of the fruit. Higher adhesion ensures longer durability of the film on the

surface of the fruit. The water solubility of a coating is another basic factor

that needs to be considered.

Chitosan form films that have good physical and barrier properties.

These properties can be improved or changed using additives such as

plasticizers. Such substances have the ability of modifying the mechanical

properties of the coatings by combining with the main components of films

and interspersing between polymer chains, moving the chains apart and

reducing the rigidity of the structures. The compounds most often used as

plasticizers in the formulation of edible coatings for cut-fruits are: glycerol

and polyethylene glycol (Guilbert and Biquet, 1996). Water may play the

role of plasticizer in hydrophilic coatings (Cisneros-Zeballos and Krochta,

2002).Films with improved or different physical (adherence, transparency),

34

mechanical and barrier properties are needed for varied type of

applications.

Argaiz (2004) determined the mechanical (hardness and

elongation), physical (color) and barrier (oxygen, water vapor and carbon

dioxide permeance) properties of 1 % chitosan films fabricated with four

plasticizers (lauric, miristic, palmitic acid and olive oil) at two

concentrations (0.3 and 0.6 %) and two thicknesses (38.1 and 76.2 µm).

In general, the film thickness increased with decrease in plasticizer

concentration and, water vapor and carbon dioxide permeability

decreased. Oxygen permeability values ranged from 0.023 to 0.704

cm3/cm2 min. Chitosan films with 0.3 % palmitic acid and 38.1µm thick

were the least permeable to oxygen. Gas permeability showed a direct

relationship with plasticizer concentration.

Physical, mechanical and barrier properties of chitosan films can be

controlled choosing the appropriate plasticizer, its concentration, and

thickness, since these parameters affect significantly the properties of

chitosan films. Chitosan films have moderate water vapor permeability and

exhibit good barrier properties to permeation of oxygen (Cervera et al,

2004). Caner et al. (1998) studied the effects of acid concentrations,

plasticizer concentrations, and storage time on the mechanical and

35

permeation properties of chitosan films. Tab. 2.4 shows a list of

applications and functions of a few edible coatings.

Barrier properties of edible films include water vapor permeability,

gas permeability, volatile permeability and solute permeability. Most edible

films are characterized by gas and water vapor permeability because

these properties affect the quality of the enclosed food. O2 permeation

occurs by gaseous diffusion while water vapor transmission involves both

water vapor sorption and diffusion. Polysaccharide films like chitosan

which are more hydrophilic tend to be less permeable to gases at low

relative humidity. Their permeability changes with temperature and relative

humidity (Guilbert, 1986). RH affected the hydration status of the film and

thus its permeability.

Tab. 2.4: Edible coating applications and functions

Type of edible coating Function Reference

Polysaccharide coatings

I Cellulose Carboxymethyl cellulose

Bananas Apples

Fresh fruits and

O2 and CO2 barrier

"

"

Banks 1984 Banks 1985; Drake et al., 1987 Lowings and Curts 1982

36

vegetables Freshly-cut celery Pears Tomatoes Oranges

Moisture barrier O2 and CO2 barrier

" "

Mason 1969 Meheriuk and Lau 1988 Nisperos & Baldwin 1988Nisperos-Carriedo et al., 1990

II Starch Dextrins (starch hydrolysates)

Freshly sliced apples

O2 barrier

Murray and Luft 1973

III Seaweed Extracts Carrageenan

Cut grape-fruit halves

Moisture barrier

Bryan 1972

IV Chitin/Chitosan

Apples, pears, peaches, plums

Fresh strawberries

Fresh cucumbers, bell peppers

O2 and CO2 barrier

Post harvest decay control

Post harvest decay control

Davies et al., 1989; Elson and Hayes 1985

El Ghaouth et al., 1991

El Ghaouth et al., 1991

Protein Coatings

I. Corn Zein Tomatoes

Moisture and O2 barrier

Park et al., 1994

37

Type of edible coating Function Reference

II Casein a) Casein-acetylated monoglyceride

Zucchini

Apples and celery sticks

b) Casein-stearic acid, beeswax, or acetylated monoglyceride

Peeled carrots

Moisture barrier

Moisture barrier

Moisture retention

Avena-Bustillos, Krochta, et al., 1994

Avena-Bustillos et al., 1997

Avena-Bustillos et al., 1993, Avena-Bustillos, Cisneros-Zevallos et al., 1994

Krochta and de Mulder-Johnston 1997

The properties of the film (functional, nutritional, organoleptic, and

mechanical) can be improved by the use of additives such as antibrowning

agents, preservatives, firming agents, plasticizers, nutraceuticals, volatile

precursors, flavors, and colors, widening the usefulness of coatings for

minimally processed fruits. Some of these additives are more effective on

food when applied as part of an edible coating than when applied as

aqueous solutions by spraying or dipping, since the coating can maintain

additives on the surface of the food for longer time (Baldwin et al., 1996).

38

Some additives that have been used on coated cut-fruits include calcium

chloride to inhibit loss of firmness, ascorbic acid to decrease browning

rate, and potassium sorbate and benzoic acid to inhibit microbial growth.

Feeding trials have recently demonstrated that chitosan is non-toxic

and biologically safe (Zhang and Quantick, 1998; Rao and Sharma, 1997;

Jiang and Li, 2001). In Japan chitosan and its derivatives are approved as

functional food ingredients. It has received the GRAS (Generally

Recognized As Safe) status for use in animal foods and purification of

potable water (Knorr, 1986). Though it has been approved in Japan and

Canada for various food applications, FDA has not yet approved chitosan

for edible use in the USA.

Chitosan films have been tried on a wide variety of fruits and

vegetables like apples, pears, strawberry, tomatoes, litchi, mango,

banana, small berry fruits, breadfruit, mushrooms, bell pepper, cucumber,

carrots, and avocado (Kittur et al., 2001, Buchner et al., 2003, El Ghouth

et al., 1991, Zhang and Quantick, 1998, Worrel, 2001, Durango et al.,

2006, Zhang and Quantick, 1997). These results are mainly attributed to

decreased respiration rate, delayed ripening due to the reduction of

ethylene and carbon dioxide evolution, and inhibition of fungal

39

development. It has been applied on dairy and sea foods too (Simpson et

al., 1997).

There are some disadvantages in the use of edible films. These

could be overcome by suitable selection of the type and thickness of the

coating and by treating sound fruits of right maturity and avoiding storage

of coated fruits at high temperature (Park et al.,1994). Thick coatings

could restrict the respiratory gas exchange, causing the product to

accumulate high levels of ethanol and to develop off- flavors (El Ghaouth

et al., 1992). Poor water vapor barrier properties of the coatings may

result in weight or moisture loss of the product, but it could also prevent

water vapor condensation, which could be a potential source of microbial

spoilage for fruit and vegetable packaging (Ben-Yehoshua, 1985).

Films that have good gas barrier properties could cause anaerobic

respiration and interfere with normal ripening. The film should allow a

certain amount of oxygen permeation through the coating or film in order

to avoid anaerobic conditions. The functional characteristics required for

the coating depend on the product matrix (low to high moisture content)

and deterioration process to which the product is subject (Guilbert et al.,

1996).

40

Chitosan films with natural preservatives

Incorporation of natural antimicrobial agents into biodegradable

films would be an ideal solution to the environmental and food safety

concerns. Lately, food scientists have been investigating this aspect and

are coming up with a host of new biopreservatives that may soon be

added to commercially prepared foods or incorporated into food packaging

(Raloff, 1998). These consumer-oriented trends have led to a renewed

interest in the development of biopreservatives for extending the shelf-life

and maintaining the safety of foods.

Although the antimicrobial properties of many compounds from

plant, animal and microbial sources have been reported, their potential for

use as natural food preservatives has not been fully exploited (Roller,

1995). As with other MAP, edible films can create a very low O2

environment where anaerobic pathogens such as C. botulinum may thrive.

Antimicrobial compounds that can be incorporated into the coating are

also being investigated (Farber, 2003).

Multicomponent edible films and coatings are being formulated

leading to innovative applications for the benefit of the food industry. Park

et al. (2002) investigated the feasibility of developing a chitosan based

antimicrobial packaging film through the incorporation of chitosan into low

41

density polyethylene (LDPE) films. The controlled release of chitosan from

film matrix seems to effectively inhibit bacterial growth if the amount of

chitosan in film matrix is sufficient to inhibit the recovery of microbial

growth.

Antimicrobial agents have been incorporated into polymers used in

packaging by solvent compounding or in the melt. In solvent compounding

both the antimicrobial and the polymer need to be soluble in the same

solvent (Appendini and Hotchkiss, 2002, Park et al., 2004).

Chi et al. (2003) report that microbial growth is predominant at the

surface of many food products. Incorporation of essential oils (EOs) in

chitosan coatings can protect ready-to-eat food from pathogen and

spoilage microorganisms. Antimicrobial effects of chitosan films enriched

with essential oils against Escherichia coli O157:H7 and Listeria

monocytogenes were evaluated by disk diffusion method at 35°C. The

films were prepared from solutions containing 1% chitosan, 1% acetic

acid, 0.5% Tween20, and 1 to 4% EO (anise, basil, coriander, and

oregano). They found that L. monocytogenes was more sensitive to all

essential oils, applied alone or incorporated in chitosan films, than E. coli.

Inhibition activity toward both bacteria was strongest with oregano EO,

followed by basil and coriander, and the weakest with anise EO. Their

42

study indicates that chitosan-essential oil films have potential in ensuring

food safety due to local application of antimicrobials and reduced losses of

active volatiles.

2.4.2.3 Lysozyme as biopreservative

Lysozyme is a popular biopreservative that is a lytic enzyme. It is

obtained from egg white and is heat stable at pH <5.3. Gram positive

bacteria are very sensitive to this enzyme. Plasticizers used in the film

forming solutions stabilize the enzyme (Cagri and Ryser, 2004). Due to its

good stability it is used more extensively in food preservation due to its

wide range of pH and temperature.

Park et al. (2004) and Song et al. (2002) have reported that the

antimicrobial spectrum of lysozyme can be extended to Gram negative

bacteria when combined with chitosan and have confirmed the

antimicrobial activity of the film. It possesses enzymatic activity against the

beta 1–4 glycosidic linkages between N-acetylmuramic acid and N-

acetylglucosamine found in peptidoglycan. Peptidoglycan is the major

component of the cell wall MRS broth and reconstituted skim milk, and

inactivation by proteolytic enzymes in buffer. According to Proctor and

Cunningham (1988) the lysozyme attaches itself to the amino sugar

residues of the polysaccharide chain of the bacterial cell wall, bringing

43

about a distortion and cleavage which results in a punctured cell wall. It is

used mostly in Japan in meat and fish products and also on packaging

films. Studies have shown that the antimicrobial spectrum of lysozyme can

be enhanced when it is used with other substances like hydrogen peroxide

and ascorbic acid (Miller 1969), EDTA (Padgett et al., 1998), caffeic acid

(Valenta et al., 1998), alginate and carrageenan (Cha et al., 2002) and

chitosan (Song et al., 2002) and its antimicrobial activity has been

evaluated.

Research on the efficacy of lactoferrin and lysozyme incorporated

into casein or zein films to keep out E.coli revealed that the casein films

exhibited good mechanical properties and significantly inhibited the growth

of E.coli. The inhibitory effects were dependent on the levels of

antimicrobials. The result implied that lactoferrin and lysozyme could be

used in the edible films to enhance food safety (Oh et al., 2003). A study

by Subburathinam et al. (2005) demonstrated the synergistic antimicrobial

effects of crawfish chitosan and lactoferrin against L. monocytogenes and

E. coli 0157:H7.

Although lysozyme exerts antimicrobial effects only on Gram-

positive bacteria, HMC conjugation extended the antimicrobial activity to

Gram-negative bacteria and greatly improved its emulsifying properties.

44

No cell toxicity has been reported in egg white–polysaccharide conjugate

when tested with mammalian cells, and the protein structure in the

conjugate was kept in the native form (Nakamura et al., 1992). Therefore,

HMC–lysozyme conjugate can be potentially used in formulated food or

drug systems since it possessed novel bifunctional properties even under

acidic conditions.

Lysozyme is an approved additive used in dairy and meat

products. The effect of lysozyme and salts on the growth of the shrimp

microflora was investigated. It was found that lysozyme at concentrations

up to 150 µg/ml could retard microbial growth in nutrient broth at 28°C. No

growth was discernible using concentrations of 50µg/ml lysozyme and

0.02% Na2EDTA, either in nutrient broth or in 2% shrimp homogenate.

Since the antimicrobial or antioxidant can be incorporated and applied

directly to the surface of the product, only small quantities are required.

Following incorporation into the films, both nisin and lysozyme maintained

their antimicrobial capacity against the indicator organisms Lactobacillus

plantarum and E. coli, which was augmented by the addition of a chelating

agent such as EDTA (Padgett et al., 1998).

Edible film made of shellfish chitosan and lysozyme can bring about

lysis by changing the integrity of the microbial cell membrane. They may

45

be suitable for packaging for ready-to-eat meats such as hot dogs,

sausage and luncheon meat; packing films for cheese slices, blocks and

sticks; and coatings for sliced fruits and vegetables that are highly

perishable (Herring, 2004).

Park et al. (2004) studied the antimicrobial activity and film

properties of chitosan-lysozyme films. The physical properties are shown

in Tab 2.5. They demonstrated the enhanced antimicrobial activity of

these composite films. Lysozyme was released in a controlled manner and

retained its antimicrobial function against both Gram-positive and gram-

negative bacteria. There was good biocompatibility between the two

components of the films and lysozyme was homogenously distributed

throughout the chitosan matrix. Therefore this film has been suggested for

use on highly perishables fruits and vegetables.

Since tomatoes are available in plenty during seasons, application

of edible films may help to extend its shelf life, till it is packed off to a

distant market or held for a few more days for a better price. Chitosan-

lysozyme films are claimed to be effective against fungi, Gram-positive

and Gram-negative bacteria (Park et al., 2004); hence, this study

investigates the efficacy of these films in extending the shelf life of

tomatoes.

46

Tab. 2.5: Properties of chitosan (ch) – lysozyme (ly) films

Film type Density

(g/ml)

Moisture

(%)

Thickness

(µm)

Tensile strength

MPa

Elongation

(%)

Water vapor

permeability (g mm/m2)

2% ch + 0% ly

1.34 23.6 74.9 17.4 60.3 177.2

2% ch + 20% ly

1.3 21.7 70.8 14.4 53.8 157.4

2% ch + 60% ly

1.3 19.0 73.0 9.5 39.3 160.0

2% ch+100%

ly 1.31 18.8 69.9 7.4 29.1 166.2

Adapted from Park et al. (2004)

This research work has been designed to study the effect of

chitosan-lysozyme coatings on the respiration, shelf life and quality of

tomatoes stored in two different storage conditions: ambient and optimal

conditions. Each of them has been studied separately.

47

Chapter III

Respiration and Quality Changes in Tomatoes Coated with

Chitosan – Lysozyme Films during Ambient Storage Conditions

3.1 Introduction

Post harvest technology is of great importance in preventing both

qualitative and quantitative losses in fruits and vegetables, which is high in

developing countries. Biological processes like respiration continue even

after harvest till its energy reserves can sustain it. Handling of the produce

in a suitable way to prolong its life is of great concern to the food industry.

Storage conditions play a very important role in influencing the rate at

which these processes take place.

Respiration is a process by which oxygen (O2) is used and carbon

dioxide (CO2) is produced. Under normal atmospheric conditions, aerobic

respiration takes place, whereby glucose is oxidized to CO2. The pace of

deterioration of harvested commodities is generally, related to the

respiration rate (Fallik and Aharoni, 2004). The respiration rate alters

during a commodity’s natural process of ripening, maturity and

senescence. The ratio of CO2 produced to O2 consumed, known as the

respiratory quotient (RQ), is normally assumed to be one, but can range

from 0.7 to 1.3 depending on the metabolic substrate utilized. Ethylene

48

production rates by fresh horticultural commodities are reduced during

storage at low temperature, by reduced O2 levels, and elevated CO2 levels

around the commodity (Aharoni, 2004a).

Application of edible coating on the produce is known to extend its

storage life by reducing respiration rate. It has received much attention of

late not only due to the environmental consciousness and consumer

demand for high quality convenience food with long shelf life, but also for

creating market outlets for traditional and new agricultural crops which are

the sources of film forming ingredients (Wu et al., 2002; Krochta. and De

Mulder-Johnston, 1997). Though there are many formulations developed it

is yet to be widely used by the industry. Each commodity has very typical

post harvest behavior and the film is formulated to suit this requirement.

Besides, the storage conditions will also affect the performance of the film

in extending the shelf life of the produce.

The important barrier properties of edible films include water vapor

and oxygen permeabilities (Guilbert et al., 1996). Highly polar polymers

like polysaccharides show low gas permeability at low relative humidity

(RH). They are sensitive to humidity changes- cracking at low RH and

swelling at high RH, thus affecting their barrier function. The oxygen

transmission and moisture uptake by the film is affected by RH. Studies on

49

composite films by Kester and Fennema (1986) have also shown the

differences in the performance of the films due to temperature variations.

The moisture sorption characteristics of the films are altered with changes

in temperature.

Films for packaging are selected based on their specific

permeability characteristics, and physical changes take place in these

characteristics as a function of time, temperature, and humidity. The

reduced O2 and increased CO2 that results from tissue respiration creates

gradients across the film barrier that provides the driving force for gas

movement into and out of the package. The levels of O2 and CO2 within a

package depend on the interaction between commodity respiration and

the permeability properties of the film (Beaudry et al., 1992; Kader et al.,

1997).

Transpiration occurs due to morphological and anatomical

changes, surface area exposed, surface injuries and maturity stage and

also RH, air movement and atmospheric pressure. This physical process

can be controlled by coating with waxes and plastic films as barriers

between the produce and the environment, besides manipulating RH,

temperature and air circulation (Aharoni, 2004b).

50

Some edible coatings have been found promising in extending the

storage life of horticultural crops. Tomato is a very widely used nutritious

fruit that is always in high demand. Limited shelf life is a major problem in

marketing tomatoes. Edible coatings create a modified atmosphere within

the fruit and thus help to preserve it for a longer time. Studies on edible

film application of fruits and vegetables are done under certain conditions

of storage. Changes in temperature or RH will bring about differences in

the function of the film as mentioned above. Hence performance and

suitability of a film will also depend on the conditions of the atmosphere

around the produce.

3.2 Objectives

The objectives of this study were to determine 1) the respiration

rate of the coated and uncoated fruits when stored under ambient

conditions of 220C and 35% RH and 2) the changes in quality of tomatoes

during storage.

3.3 Materials and methods

Tomatoes were purchased in their turning stage from the local

market. Fruits with uniform size color and free from damage and fungal

infection were washed twice in water and then held in chlorinated water for

5 min. They were then drained and surface dried in air. They were

51

separated into lots of 18 fruits for each treatment on a completely

randomized design. Three replicates were used.

The chitosan, lysozyme and other chemicals were purchased from

Sigma Chemical Co, USA.

Film forming solutions

Using crab shell Chitosan (85% deacetylated) (Sigma) 1 and 2%

solutions were prepared using 0.6% acetic acid, adding 25% glycerol (w/w

chitosan) as plasticizer. A 10% lysozyme stock solution was prepared in

distilled water adding 25% glycerol (w/w lysozyme). The film forming

solutions were made by mixing lysozyme in 1 or 2% chitosan solution so

as to give 0, 20, 60, 100% lysozyme (percent dry weight lysozyme per dry

weight of chitosan) as suggested by Park et al. (2004). Each of the

solutions was thoroughly mixed, filtered and the pH was adjusted to 5.2

using 1N sodium hydroxide.

This resulted in a set of treatments as listed below: (% Lysozyme is

expressed as a ratio of dry wt. of lysozyme per dry wt. chitosan)

1. Control - Uncoated

2. C1 - 1% chitosan + no lysozyme

3. C1L0.5 - 1% chitosan + 20% lysozyme

4. C1L1 - 1% chitosan + 60% lysozyme

52


6. C2 - 2% chitosan + no lysozyme

7. C2L0.5 - 2% chitosan + 20% lysozyme



Tomatoes were dipped individually in the film forming solutions for

10 s, drained and dried in open air at room temperature for 6 h. The

uncoated tomatoes served as control. They were placed in paper bags

and held at ambient conditions of an average temperature of 220 C and

RH of 35%.

Respiration

Respiration rate was determined by sealing one fruit in a 1L glass

bottle for about 1-4h when three 0.5ml samples of gas at headspace were

drawn with gas-tight hyperdermic syringes and analysed for CO2 and O2

using gas chromatography (SRI Gas Chromatograph, model 8610A). To

analyse the internal atmosphere of the fruits three 0.5ml samples of gas

were drawn from the core tissue of the stem scar as suggested by Pharr

and Kattan (1971). They were analysed as described above. For each

treatment three single fruit replicates were analysed on 2, 5 and 10 days

of storage.

53

Quality analyses

Weight loss of the fruits was determined using an electronic

balance. Color was measured as L*, a* and b* color coordinates using

Minolta chromameter (model CR 300X, Minolta Camera Co. Ltd., Japan)

and the hue angle was calculated as given by Park et al., (1994). The

instrument was calibrated against a standard white color plate (Y = 93.9, x

= 0.313, y = 0.321).

The color was measured at three points on the circumference of the

fruit and mean values calculated (Batu, 2003). As suggested by Mitcham

et al., (1996) total soluble solids (TSS) was determined using a

refractometer (Fisher Scientific, China). Titratable acidity was found by

titrating the fruit pulp to a pH of 8.1 with 0.1 N sodium hydroxide using a

digital pH meter (Accumet 25, Fischer Scientific, NY, USA). The pH was

also determined using the same pH meter. Firmness was measured using

Instron Universal Testing Machine (model 4502, Instron Instruments

Corp., Massachusetts, USA) set to operate at a crosshead speed of 25

mm/min, a force of 50 N and using a 5 mm flat steel probe. The

penetration force (N) required to penetrate through the skin was measured

on the equatorial plane by cutting the fruit longitudinally into half (Batu and

Thompson 1998).

54

The mass, color and visual quality of tomatoes were evaluated for

all the fruits weekly and the destructive tests like TSS, pH and acidity were

determined at the start and at the end of the study period.

Visual quality

The visual quality of the tomatoes was evaluated by subjective

method using a rating scale of 5 points (Aguayo et al., 2004, Ke et al.,

1991 and Talukder et al., 2003). The parameters for evaluation of the

quality of tomatoes are shown below:

Visual Appearance 1 very poor 2 Poor 3 Fair 4 Good 5 Very good

Defects* 1 >50%

2 25-50% 3 10-25% 4 10% 5 None

Shrinkage 1 >50%

2 50% 3 25% 4 10% 5 None

*Defects include microbial spoilage, discoloration, pitting and softening

55

Data analyses

The data was subjected to analysis of variance (ANOVA) and

Duncan’s multiple comparison test of means using the software XL STAT

2006. The results were used to determine the Least Significant

Differences (LSD) amongst treatments at a significance level of 0.05.

3.4 Results and Discussion

CO2 production

10

15

20

25

30

35

0 2 4 6 8 10 12

Duration, days

CO

pro

duct

ion

rate

, mg/

kg/h

r

ControlC1C2C1L2C2L2

LSD(0.05)= 4.39

2

Fig. 3.1: CO2 production of tomatoes stored at ambient conditions

Application of chitosan coating affected CO2 production. There was

little difference between the uncoated and the coated tomatoes, except

C2, (Fig. 3.1) at five days of storage. It fell sharply by the tenth day for

56

both the 1% chitosan treatments which were found to be different from the

control (p<0.05). There wan a steady rise in CO2 in C2 and C2L2 upto the

fifth day, and remained high till tenth day. After ten days, control samples

were significantly different from 1% chitosan treatments. The CO2

production of C2L2 was significantly higher than the control (p<0.05);

though C2 was not significantly different from control the values were

greater. The initial rise in CO2 production seen in the coated fruits could be

a stressful response to the treatment (El-Ghaouth et al., 1992). One

percent chitosan treatment was effective in reducing CO2 production which

is different from that observed by El-Ghaouth et al. (1992), who stated that

there was no difference between the one and two per cent chitosan

treatments. The above results comply with those of Cantwell (2000).

Respiratory quotient

0.000.250.500.751.001.251.501.752.002.252.502.75

0 2 4 6 8 10 12

Duration, days

RQ

ControlC1C2C1L2C2L2

LSD(0.05)= 0.24

Fig. 3.2: Respiratory quotient of tomatoes stored at ambient conditions

57

The difference in RQ was observed from 2nd day onwards. While

RQ of all coated tomatoes (Fig. 3.2) rose to a peak by 5th day and

decreased in the next five days, uncoated tomatoes showed a rise in RQ

which continued to be so upto 10 days. On the 2nd day C1 and control

were similar and the other treatments were significantly different (p<0.05).

After 5 days, there was less difference amongst the coated tomatoes,

though their RQ values were all much higher than the control (p<0.05).

This could be a response to the coating. These values fell during the next

five days indicating a more favorable reaction in the coated tomatoes. The

RQ of C1 and C1L1 fell below that of control, which was not significant. C2

and C2L2 showed significantly higher RQ (p<0.05) compared to control

and 1% chitosan treatments.

Internal CO2

In two days there were significant differences between coated and

uncoated tomatoes (p<0.05) as seen from Fig.3.3. The same trend was

observed on the fifth and tenth day. There were no significant differences

between C1 and C1L2 and between C2 and C2L2 throughout the storage

period, indicating that the addition of lysozyme did not affect internal CO2

58

production. The coatings as such brought about an increase in the internal

CO2, the highest being in the 2% chitosan treatments.

0

2

4

6

8

10

12

14

16

18

0 2 4 6 8 10 12

Duration, days

% C

O

ControlC1C2C1L2C2L2

LSD(0.05)= 0.68

2

Fig. 3.3: Internal CO2 of tomatoes stored at ambient conditions

Internal O2

0

2

4

6

8

10

12

14

16

18

20

0 2 4 6 8 10 12

Duration, days

% O

ControlC1C2C1L2C2L2

LSD(0.05)= 0.58

2

Fig. 3.4: Internal O2 of tomatoes stored at ambient conditions

59

There was a similar trend in the reduction of internal O2 (Fig. 3.4) in

all the tomatoes. The control, 1% and 2% chitosan treatments were found

to be significantly different (p<0.05) from each other throughout the

observation period. C2 and C2L2 were similar and their endogenous O2

was the lowest. There was a quick decrease till five days, thereafter the

decrease was gradual. C1 and C1L2 had higher internal O2 than 2%

chitosan. Untreated tomatoes had the highest levels of O2. These values

follow a similar pattern as obtained by El-Ghouth et al. (1992).

Park (1999) states that gas diffusivities of tomato exocarp and

pericarp and stem scar increased as ripening takes place. Low O2 and

elevated CO2 can significantly reduce the rates of ripening and

senescence primarily by reducing the ethylene synthesis and sensitivity to

it. Changes in respiration and starch, sugars, chlorophyll, and cell wall

constituents during ripening and/or senescence can be reduced or

arrested, by eliminating ethylene action through the use of low O2 and high

CO2 atmospheres. During ripening there is degradation of pectin that

changes the diffusivities of gases through the skin of the tomato. Lower O2

limit increased with temperature. Lower O2 limits vary from 0.15% to 5%

and for tomato it is 2% at optimal storage conditions. The film around the

60

fruit acts as a barrier limiting exchange of moisture and gases, depending

on its thickness and other properties.

Mass loss

Mass loss occurs in fruits due to respiration and by transpiration

due to the water vapor pressure difference between the fruit and the

atmosphere (low relative humidity). Coating fruits with a film checks

moisture loss keeping them fresh during storage.

0

2

4

6

8

10

12

Control C1 C1L0.5 C1L1 C1L2 C2 C2L0.5 C2L1 C2L2

Treatment

Mas

s Lo

ss %

7d14d21d

LSD(0.05)= 0.44

Fig. 3.5: Changes in mean mass loss of tomatoes during storage

As shown in Fig. 3.5 there is a considerable increase in mass loss

over the storage period in all the treatments. The untreated tomatoes

show a significantly higher mass loss (p<0.05) on the 7th, 14th and the 21st

day compared to the treated tomatoes.

61

Amongst the coated tomatoes there is not much of variation seen

during the first 7 days, but by the second and third week the mass loss in

C1L2 treatment rises much slowly than the other treatments. Having the

lowest mass loss C1L2 showed a statistically significant difference

(p<0.05) from the control.

Overall comparison shows that there was no statistical difference

between the control and C1, C1L1 and C2 but the control was significantly

different from C1L2, C2L0.5, C2L1 and C2L2 (p<0.05). There was no

considerable difference in mass loss between 1% and 2% chitosan

treatments though mass loss was higher in C2 and C2L2. The chitosan

concentration is one of the factors determining film thickness which in turn

affects moisture and gas permeability due to its specific physical and

mechanical properties. The treatments with C1L2 and C2L1 seem to have

better moisture barrier properties. The difference between treatments

depends on the reaction of the fruit to the properties of the film and the

storage temperature and humidity.

Mass loss values being slightly higher in the 2% chitosan coating

could be due to the adverse reaction in the fruit due to excessive

thickness of the film as stated by Park et al. (1994). They have reported a

mass loss of 6 to 7% in tomatoes coated with corn-zein film after storage

62

at 21℃ for 14 days. Increased mass loss in thickly coated tomatoes is

reported to be due to loss of sugars, heat generation and production of

end products from anaerobic fermentation. Chitosan films are said to be

more permeable to moisture than other polysaccharide films, thus making

them suitable for fruits and vegetables (Miranda et al., 2004). Tasdelen

and Bayindirli (1998) have also observed that sucrose polyester coating of

tomatoes stored at 23℃ reduced mass loss but it was not significantly

different from the uncoated.

Color

Color is a very important determinant of quality and consumer

acceptability, especially with respect to tomatoes (Aked, 2000). During

ripening the green chlorophyll pigment is degraded and there is

accumulation of carotenoids giving the red color to the ripe tomato.

L* values

Tab. 3.1: Mean L* values of tomatoes during storage at ambient conditions

Means with same letters are not significant (P<0.05)

Treatment 0d 7d 14d 21d Control 50.95±0.00 a 39.77±0.00 c 36.04±0.00 b 33.02±0.00 c C1 50.50±1.13 a 42.37±1.20 abc 42.81±0.88 ab 41.79±1.36 ab C1L0.5 51.08±1.24 a 41.84±1.20 abc 43.63±0.79 a 41.36±0.96 ab C1L1 52.58±1.13 a 40.68±1.35 bc 42.71±0.79 ab 42.83±0.96 ab C1L2 50.30±1.13 a 41.82±1.35 abc 43.87±0.88 a 41.89±0.96 ab C2 50.63±1.13 a 45.47±1.35 abc 43.49±0.79 ab 43.38±0.96 ab C2L0.5 51.19±1.24 a 45.43±1.20 ab 43.49±0.79 ab 43.03±0.96 ab C2L1 50.94±1.13 a 45.55±1.20 ab 43.21±0.79 ab 43.67±0.96 ab C2L2 50.51±1.13 a 43.76±1.20 a 42.48±0.88 ab 41.85±1.07 ab

63

The L* values decreased (Tab. 3.1) during the first 7 days and less

change was seen thereafter. The change was more in the control which

was significantly different (p<0.05) from C2L0.5, C2L1 and C2L2. On 14th

day C1L0.5 and C1L2 were significantly different (p<0.05) from the

uncoated tomatoes. By the third week the control was considerably

different from the treated ones (p<0.05). The L*values for 2% chitosan

treatments showed slightly higher values than 1% chitosan treatments.

According to Camelo and Gomez (2004) L* values do not change until

after the turning stage of maturation when the fruits begin to turn from pink

to red color. Higher L* value indicates darker color.

Hue values

Tab. 3.2: Mean hue values of tomatoes during storage at ambient conditions

Means with same letters are not significant (P<0.05)

Tab. 3.2 shows the changes in hue values that indicate color

change from green to red. Normally this value decreases during ripening.

Treatment 0d 7d 14d 21d Control 82.47±0.00 a 50.98±0.00 c 34.80±0.00 c 30.96±0.00 b C1 82.76±1.30 a 62.20±1.30 b 41.35±4.62 a 33.84±6.39 a C1L0.5 83.50±1.42 a 64.08±1.42 ab 44.55±4.13 ab 35.33±4.52 a C1L1 81.42±1.30 a 62.43±1.30 b 42.47±4.13 b 35.39±4.52 a C1L2 83.74±1.30 a 61.85±1.30 b 43.77±4.62 ab 34.36±4.52 a C2 82.67±1.30 a 75.59±1.30 a 45.37±4.13 ab 38.57±4.52 a C2L0.5 82.41±1.42 a 72.92±1.42 a 48.00±4.13 ab 38.68±4.52 a C2L1 82.76±1.30 a 68.42±1.30 a 48.31±4.13 b 37.55±4.52 a C2L2 82.15±1.30 a 69.84±1.30 a 47.18±4.62 b 38.25±5.05 a

64

The hue value of the uncoated tomatoes was significantly different

(p<0.05) from the coated ones throughout the storage period. By the first

week there were significant differences between 1% and 2% chitosan

treatments, except C1L0.5. By 21 days there was less difference, though

tomatoes with 2% chitosan treatment continued to show less ripening. On

21st day there was a significant difference between the control and coated

tomatoes (p<0.05) and the treated tomatoes did show higher values.

As the fruits ripened during storage the hue values decreased.

Between treatments, it was seen that two per cent chitosan coating

showed a marginal delay in ripening compared to the one per cent

chitosan treatment. These observations agree with those observed by

Park et al. (1994) and Tasdelen and Bayindirli et al. (1998). Park et al.

(1994) state that ripening was delayed in tomatoes even when coating

was applied at the pink stage of maturity. El-Ghaouth et al. (1992)

observed delayed ripening in both 1% and 2% chitosan coating during a

storage period of 25 days at 20℃.

Film coating of fruits helps to delay the process of ripening as seen

from the higher hue values in coated tomatoes. Two percent chitosan

treatment was more effective in delaying ripening. This could be due to the

65

check on respiration brought about by the increased CO2 production

around the fruit (Shewfelt, 1986).

Visual Quality

Appearance

Tomatoes are evaluated for quality first by their appearance.

Consumers prefer tomatoes with no blemishes and which are clean, fresh,

shiny and bright colored.

0

1

2

3

4

5

6


Treatment

App

eara

nce

(sco

res)

0d7d14d21d

LSD(0.05)= 0.44

Fig. 3.6 : Mean scores for Appearance of tomatoes during

storage at ambient conditions Fig. 3.6 shows that the mean score for appearance began to fall

appreciably from the second week of storage. During the first week all

except C2L0.5 scored well close to maximum. During the second week

the uncoated tomatoes, C2 and C2L0.5 scored significantly lower scores

(p<0.05) than C1, C1L0.5, C1L1 and C1L2 treatments. By the third week it

66

was found that C1L0.5, C1L1 and C1L2 treatments continued to score

better than the other treatments (p<0.05). Most of these tomatoes were

shiny and appealing with no scars even after three weeks storage. Of all

the treatments C1L0.5, C1L1 and C1L2 coatings gave good scores for

appearance which was significant (p<0.05). This indicated that 1%

chitosan was more compatible to preserve the freshness of the tomatoes.

Defects

0

1

2

3

4

5

6


Treatment

Dis

ease

/Def

ects

0d7d14d21d

LSD(0.05)= 0.44

Fig. 3.7: Mean scores for Defects in tomatoes during storage at ambient

conditions

Fig. 3.7. shows the scores for defects like discoloration, decay,

pitting and softening, higher the score lesser the defects. By the first week

C2 and C2L0.5 treatments resulted in significantly low scores (p<0.05)

67

compared with all other films. This trend continued during the second

week when C2 and C2L0.5 scored significantly low indicating more

defects. On the 21st day treatments C1L0.5, C1L1, C1L2 and C2L1 were

found to score better than the rest (p<0.05). On the whole C1L1 scored

the best. The 1% chitosan samples scored better than the rest, though the

means were not statistically different. Tissue softening with water

accumulation was more commonly seen in 2% chitosan treatments which

indicate its unsuitability. The addition of lysozyme did not seem to

influence the quality of the fruits as there was no significant difference

between tomatoes with lysozyme and without lysozyme. There was much

less pitting and blemishes in the treated tomatoes compared to the

control. Even after 21 days of storage fungal spoilage was minimum in the

treated tomatoes. The protective effect of the film and the low RH might

have helped maintain the quality of tomatoes.

Shrinkage

Shrinkage on the surface of a fruit is a result of moisture loss during

storage due to low relative humidity. Thick films also show wrinkling during

storage due to their physical properties and the plasticizer used (Miranda

et al., 2004). As stated by Wu et al. (2002) temperature and RH play a

very important role in deciding the barrier properties of edible films.

68

0

1

2

3

4

5

6


Treatment

Shrin

kage

(sco

res)

0d

7d

14d

21d

LSD(0.05)= 0.43

Fig. 3.8: Mean scores for Shrinkage of tomatoes during storage at ambient

conditions

Fig. 3.8 shows that shrinkage was noticed by the first week in C1,

C1L2, C2, C2L0.5 and C2L2. Significant increase (p<0.05) in signs of

shrinkage was seen on the 14th day in the untreated and all the treated

tomatoes except C1L0.5, C1L1 and C1L2. By the third week shrinkage

was observed in all the tomatoes, with control and C2L2 treatment scoring

very low (p<0.05). Upto two weeks there was no shrinkage seen in C1L1.

The concentration and the composition of the films seem to influence

shrinkage. In the present study mass loss did not differ between 1% and

2% chitosan treatments. Shrinkage could be due to the response of the

film to the low RH.

69

Firmness

Tab. 3.3: Mean final values (in N) for firmness of tomatoes during storage at ambient conditions

Initial firmness was 19.2N. Means with same letters are not significant (P<0.05)

Firmness of the tomatoes was better preserved by the application

of the films as seen in Tab. 3.3. It decreased in both coated and uncoated

tomatoes from the initial value of 19.22 N. At the end of three weeks

storage all the tomatoes were ripe. No significant difference was observed

between the treated and the untreated tomatoes, though the uncoated

tomatoes were least firm. There was a small variation in firmness between

1% and 2% chitosan treatments. This is an important factor indicating the

internal freshness of tomatoes. Reduction of respiration and decrease in

water loss may result in retention of firmness during storage. Use of

appropriate coating could contribute to this (Tasdelen and Bayindirli,

1998). The respiration and O2 consumption of coated tomatoes were lower

than those of noncoated tomatoes (Park et al., 1992). Progressive loss in

Treatment 21days Control 6.68±0.00 a C1 8.18±0.24 a C1L0.5 8.70±0.28 a C1L1 9.74±0.28 a C1L2 8.69±0.28 a C2 9.62±0.28 a C2L0.5 9.90±0.28 a C2L1 10.25±0.28a C2L2 10.32±0.27a

70

firmness during ripening is the result of transformation of protopectin to

pectin by enzymes.

pH

4.32

4.34

4.36

4.38

4.40

4.42

4.44

4.46

4.48

Treatment

pH


21d0d

LSD(0.05)= 0.13

Fig. 3.9: Mean pH of tomatoes during storage at ambient conditions

pH varied from the initial value during the 21day storage period. pH

increased in all the tomatoes, maximum being in the control (Fig. 3.9).

Though not statistically significant, there was a marked difference in the

pH of coated tomatoes from the uncoated, with 2% chitosan treatments

showing higher values.

71

Titratable Acidity (TA)

0.350.370.390.410.430.450.470.49

Treatment

TA %


21d0d

LSD(0.05)= 0.046

Fig. 3.10 : Mean Titratable Acidity of tomatoes during storage at ambient

conditions

There was a significant decrease (p<0.05) in acidity in the uncoated

tomatoes (Fig. 3.10). The coating brought about less decrease in acidity in

the treated tomatoes, especially in the 1% group. In the 2% chitosan

coating there were very marginal changes indicating a greater influence

on respiration. This shows that 1% chitosan does not completely inhibit

metabolic changes in the fruits though the rate of change was slow.

Generally acidity decreases with ripening as the organic acids get

metabolized (Richard and Hobson, 1987).

72

Total Soluble Solids (TSS)

3.03.13.23.33.43.53.63.73.83.94.0

Treatment

TSS

%


21d0d

LSD(0.05) = 0.31

Fig. 3.11: Mean Total Soluble Solids (%) of tomatoes during storage at

ambient conditions

During the course of ripening at ambient conditions the total soluble

solids increased in all the samples (Fig.3.11). There was no significant

difference between the TSS of uncoated and coated samples, though

C1L1 showed a maximum value of 3.9 % after 21 days. Between 1% and

2% chitosan treatments no appreciable differences could be observed.

TSS is an important factor to be considered with respect to consumer

acceptance. It is expected to increase during ripening and decrease

during storage (Tasdelen and Bayindirli, 1998).

73

The above observations show that chitosan coating does prevent

deteriorative changes in tomatoes to different extents depending on the

properties of the films, their concentration and composition. Lysozyme is a

lytic enzyme that can inhibit Gram-positive bacteria and when combined

with chitosan it can also target Gram-negative bacteria. The rationale of

the addition is to inhibit the growth of spoilage and pathogenic

microorganisms on the surface where contamination occurs. The added

effect of lysozyme in the films in enhancing the antimicrobial activity of the

chitosan films was not observed in this study as claimed by Park et al.,

(2004).

The tomatoes had better market quality with 1% chitosan coating

as seen in the slower changes in hue, visual quality, acidity and TSS.

Though mass loss, hue and firmness values were better in 2% chitosan

treatments, they scored less in quality. There was more surface shrinking,

incidence of softening and water accumulation seen in these fruits. This

indicates that 2% chitosan was less compatible than 1%. Nevertheless, El

Ghaouth et al. (1992) have stated that 2% chitosan coating of tomatoes

held at 200C had a better effect than 1%. Ambient conditions of storage

with low RH could have also influenced the properties of the films as

indicated by Wu et al. (2002).

74

The effect of chitosan-lysozyme film coating on tomatoes could be

seen in the reduction of internal O2 and increase in CO2. According to El

Ghaouth (1992) internal O2 levels within 12% would not cause anaerobic

respiration. They found 2% chitosan application to be effective on

tomatoes. This study indicates that 1% chitosan coating was more

favorable than 2% chitosan, which resulted in low scores for quality,

though changes in color and firmness were delayed. The levels of CO2

produced and the low internal O2 seen in 1% chitosan treatments seem to

be within the safe levels. Low O2 and elevated CO2 levels can delay the

process of ripening and senescence. It retards all the processes involved

in respiration and metabolism of starch, sugars, chlorophyll, and cell wall

constituents, thus delaying senescence.

Modification of internal atmospheres by the use of edible coatings

can increase disorders associated with high carbon dioxide or low oxygen

concentration (Park et al., 1994; Ben-Yehoshua et al., 1969; Smith et al.,

1987). The movement of O2 and CO2 is usually directly proportional to the

differences in gas concentration across the film. Safe levels of O2 and CO2

are important for package design. A lower O2 limit has been associated

with onset of fermentation and accumulation of ethanol and acetaldehyde

(Beaudry et al., 1992). Fermentation is linked to the development of off-

75

flavors and/or tissue damage. If a coating is too thick detrimental effects

can result due to an internal oxygen concentration below a desirable and

beneficial level and an associated increase in carbon dioxide

concentration above a critical tolerable level.

3.5 Conclusion

This study demonstrated that 1% chitosan coating is suitable to

extend shelf life of tomatoes stored under ambient conditions without

unfavorable changes in quality.

3.6 References

Aguayo, E., Escalona, V. and Artes, F. 2004 Quality of fresh-cut

tomato as affected by type of cut, packaging, temperature and

storage time, Eur Food Res Technol., 219:492–499

Aharoni, N. 2004a. Packaging, Modified Atmosphere (MA) and

Controlled Atmosphere (Principles and Applications.

International Research and Development course on Postharvest

Biology and Technology. The Volcani Center, Israel.

Aharoni, N. 2004b. Packaging of Fruits and Vegetables. International

Research and Development course on Postharvest Biology and

Technology. The Volcani Center, Israel.

76

Aked J., 2000 Fruits and vegetables, In: The stability and shelf-life of

food, Ed. D Kilcast and P Subramaniam, Woodhead Publishing

Limited, p249-278

Batu, A. and Thompson, A.K. 1998 Effects of modified atmospheric

packaging on postharvest qualities of pink tomatoes, Tr. J. Agr.

& Forestry, 22, 365-372

Batu, A. 2003 Temperature effects on fruit quality of mature green

tomatoes during controlled atmosphere storage, Inter. J. Food

Sci. & Nutr., 54(3), 201-208

Beaudry, R.M., Cameron, A.C. Shirazi A.and Dostal-Lange D.L. 1992.

Modified-atmosphere packaging of blueberry fruit: Effect of

temperature on package O2 and CO2. J. Amer. Soc. Hort. Sci.

117:436-441.

Camelo, A.F.L. and Gomez P.A. 2004 Comparison of color for atomato

ripening, Hortic. Bras., 22(3)jul/sept

Cantwell, M. 2000 Optimum procedures for ripening tomatoes: In

Management of fruit ripening, Postharvest handling

systems:Fruit vegetables, In Postharvest technology of

horticultural crops, Ed. Kader, A. A., UC Davis Special

publication 3311, p80

77

El Ghaouth, A., Ponnampalam, R. Castaigne, F. and Arul, J. 1992

Chitosan coating to extend the storage life of tomatoes,

Hortscience 27(9) 1016-1018

Fallik, E and Aharoni, Y. 2004. Postharvest Physiology, Pathology and

Handling of Fresh Produce. Lecture Notes. International

Research and Development course on Postharvest Biology and

Technology. The Volcani Center, Israel., 30

Guilbert, S., Gontard, N., and Gorris, L.G.M. 1996. Prolongation of the

shelf- life of perishable food products using biodegradable films

and coatings. Lebensmittel- Wissenschaft und-Technologie.

29(1):10-17.

Kader, A.A. 1997. A summary of CA requirements and

recommendations for fruits other than apples and pears. In: A.

Kader (ed) Fruits other than apples and pears. Postharvest

Hort. Series No. 17, Univ. Calif., Davis CA, CA'97 Proc. 2:1-36.

Ke, D., Rodriguez-Sinobas, L. and Kader, A A. 1991, Physiology and

Prediction of Fruit Tolerance to Low-oxygen Atmospheres,

Amer. Soc. Hort. Sci. 116(2):253-260

Kester, J. J. and Fennema, O.R. 1986 Edible films and coatings: a

review, Food Technology, December, 47-57

78

Krochta, J.M. and De Mulder-Johnston, C.L.C. 1997 Edible and

biodegradable polymer films: Challenges and opportunities,

Food Technology 51: 60 – 74.

Miranda , S. P., Garnica, O., Lara-Sagahon, V. and Cardenas, G. 2004

Water vapor permeability and mechanical properties of

chitosan, J. Chi. Chem. Soc., 49(2), 173-178

Mitcham, B., Cantwell, M. and Kader, A. 1996 Methods for Determining

Quality of Fresh Commodities Perishables Handling Newsletter,

Issue No. 85 February, p1

Park, H. J., Chinnan, M.S. and Shewfelt R.L. 1992 Coating tomatoes

with edible films: Prediction of internal O2 concentration and

effect on storage life and quality, Paper no 848Annual meeting

of Instt. of Food Technologists, New Orleans, LA, June 20-24

Park, H.J., Chinnan, M.S. and Shewfelt R.L. 1994 Edible coating

effects on storage life and quality of tomatoes, J. Food Sci.

59(3) 568-570

Park HJ. 1999. Development of advanced edible coatings for fruits.

Trends Food Sci Tech 10:254-60.

79

Pharr, D. M. and Kattan, A. A. 1971 Effects of air flow rate, storage

temperature and harvest maturity on respiration and ripening of

tomato fruits, Plant Physiology. 48:53-55

Richard, C and Hobson, G. E. 1987 Compositional changes in normal

and mutant tomato fruit during ripening and storage, J. Sci.

Food Agric., 40:245-252

Smith, S. Geeson, J. and Stow, J. 1987 Production of Modified

Atmospheres in Deciduous Fruits by the use of Films and

Coatings. Hort. Science 22:772–776.

Shewfelt, R.L. 1986. Postharvest treatment for extending shelf life of

fruits and vegetables. Food Technology 40(5):7078, 89

Talukder, S., Khalequzzaman, K.M., Khuda, S.M.K.E., Islam, M. S. and

Chowdhury, M.N.A. 2003 Prepackaging, storage losses and

physiological changes of fresh tomato as influenced by post

harvest treatments, Pakistan Journal of Biological Sciences 6

(14): 1205-1207

Tasdelen, O. and Bayindirli, L. 1998 Controlled atmosphere storage

and edible coating effects on storage life and quality of

tomatoes, J. Food Proc. Pres 22, 303-320

80

Wu, Y., Weller, C. L., Hamouz, F., Cuppet, S. and Schnepf, M. 2002

Development and application of multicomponent edible coatings

and films: a review, Adv. Food & Nutr. Res., 44, 347

81

Connecting Text

Chitosan-lysozyme films used as coating on tomatoes has an

appreciable preserving effect as observed by the respiration rate and

quality of the tomatoes held at ambient conditions. Since the properties of

these films are known to be responsive to changes in temperature and

humidity, their effect on the quality of tomatoes needs to be investigated

under optimal storage conditions of low temperature and high humidity,

which is reported in the following chapter.

82

Chapter IV

Effect of Chitosan-Lysozyme Coatings on the Quality of

Tomatoes Stored at Low Temperature and High Humidity

4.1 Introduction

Ripe tomato fruits are perishable and very liable to transport

damage that consequently leads to loss of quality and waste. This is

especially so in developing countries due to poor post harvest handling

systems and transportation of fruits and vegetables over rough roads and

uneven surfaces. For this reason fruits intended for distant markets are

usually harvested at mature-green or breaker stages so that the fruits can

endure the rigors of handling while maximizing shelf life.

Deterioration of fresh fruits and vegetables can result from

physiological breakdown owing to natural ripening processes, water loss,

temperature injury, physical damage, or microbial spoilage. All of these

factors can interact, and all are influenced by temperature. Many fruits,

vegetables, and flowers become shriveled after losing only a small

percentage of their original weight due to water loss, thus reducing its

marketability. Relative humidity, temperature of the product and its

surrounding atmosphere, and air velocity all affect water loss (Wilson et

al., 2003). A wide range of suitable technologies are available to maximize

83

the shelf-life of perishable commodities. Methods to reduce produce

respiration, water loss and the growth of pathogens are of primary

importance. Of these, refrigeration dominates as the most fundamental of

all post-harvest technologies. The storage of fresh produce can be

considerably extended if respiration can be slowed down using

refrigeration (Aked, 2000).

The post harvest life of a product is determined by its respiration

rate and is directly related to product temperature. Cooling can slow many

undesirable changes in fruits and vegetables. Most plant tissues do not

survive freezing and they are also intolerant of low temperatures well

above freezing. Rapid cooling to the lowest safe temperature is most

essential for high respiring commodities.

For maximum shelf life, fresh fruits and vegetables must be in

excellent condition. Coating fresh produce with edible films can provide an

alternative way to control and extend quality and shelf life during storage.

They can modify the internal gas composition to give the same effect as

modified atmosphere storage. Most of the research in edible coating is

aimed at developing new combinations of materials that may suit the

preservation requirements of individual food product. Operating conditions

greatly influence the properties of edible films.

84

The results of a study by de Castro et al (2005) indicated that more

severe effects resulted from earlier cold-chain breakage and that less

ripened tomato was more susceptible to abuse. There was no significant

difference between tomatoes cooled after 4-days and the tomatoes that

were cooled immediately after harvest. Cold-chain breakage showed signs

of abnormal ripening, increased weight loss and fungus development in

the fruits. Spoilage was stirred by water condensation on fruit surface

during warming.

According to Cantwell (2000), tomatoes at breaker stage have

longer storage life if stored and ripened at 15-200C. They have good visual

quality at this temperature even after ripening. Beyond 250C they begin to

soften and lose color. They are chilling sensitive at temperatures below

100C, developing symptoms of lack of uniform ripening, softness,

mealiness, decreased flavor and decay.

Chitosan protects fresh vegetables and fruits from fungal

degradation. Although the antimicrobial effect is attributed to antifungal

properties of chitosan, it acts as a barrier between the nutrients contained

in the produce and microorganisms. In addition, chitosan-based

antimicrobial films have been used to carry organic acids and spices. The

rationale for incorporating antimicrobials into the packaging is to prevent

85

surface growth in foods where a large portion of spoilage and

contamination occurs (Appendini and Hotchkiss, 2002). Since the barrier

properties of edible films differ with temperature and RH, it would be

useful to study the effect of ideal storage conditions on the efficacy of

chitosan-lysozyme films.

4.2 Objectives

This paper discusses the effect of chitosan-lysozyme coatings on

the quality of tomatoes stored at low temperature. The objectives of this

study are: i) To assess the effect of different combinations of chitosan

coatings on the shelf life of tomatoes and ii) To evaluate the quality of the

film - coated tomatoes stored at optimum conditions of low temperature

(15℃) and high humidity (90% RH).

4.3 Materials and methods

Greenhouse tomatoes of uniform size, color and free from damage

and fungal infection were purchased from the local market at the turning

stage of maturity. They were washed twice in water and then held in

chlorinated water for 5 min. They were then drained and surface dried in

air. They were separated into lots of 9 fruits for each treatment on a

randomized complete block design. Three replicates were used.

86

The chitosan, lysozyme and other chemicals were purchased from

Sigma Chemical Co, USA.

Film forming solutions

Using crab shell chitosan (85% deacetylated) 1% and 2% solutions

were prepared using 0.6% acetic acid, adding 25% glycerol (w/w chitosan)

as plasticizer. A 10% lysozyme stock solution was prepared in distilled

water adding 25% glycerol (w/w lysozyme). The film forming solutions

were made by mixing lysozyme in 1 or 2% chitosan solution so as to give

0, 20, 60, 100% lysozyme (percent dry weight lysozyme per dry weight of

chitosan) as suggested by Park et al. (2004). Each of the solutions was

thoroughly mixed, filtered and the pH was adjusted to 5.2 using 1N sodium

hydroxide.

This resulted in a set of treatments as listed below: (% Lysozyme is

expressed as a ratio of dry wt. of lysozyme per dry wt. chitosan)

1. Control Uncoated

2. C1 1% chitosan

3. C1L0.5 1% chitosan + 20% lysozyme

4. C1L1 1% chitosan + 60% lysozyme


6. C2 2% chitosan

87

7. C2L0.5 2% chitosan + 20% lysozyme



(Note: The treatments were coded for convenience based on the weight of

lysozyme used.)

Tomatoes were dipped individually in the film forming solutions for

10 s, drained and dried in open air at room temperature for 6 h. The

uncoated tomatoes served as control. The tomatoes were placed in paper

bags and held at a temperature of 15℃ and RH of 90% in an

environmental chamber for a period of 14 days.

Quality analyses

Weight loss of the fruits at the start and during storage was

determined using an electronic balance. Color was measured as L*, a*

and b* color coordinates using Minolta chromameter (model CR 300X,

Minolta Camera Co. Ltd., Japan) and hue angle was calculated as done

by Park et al., (1994). The instrument was calibrated against a standard

white color plate (Y = 93.9, x = 0.313, y = 0.321). The color was measured

at three points on the circumference of the fruit and mean values

calculated (Batu, 2003). Total soluble solids (TSS) was determined using

a refractometer (Fischer Scientific, China) as suggested by Mitcham et al.

88

(1996). Titratable acidity was found by titrating the fruit pulp to a pH of 8.1

with 0.1N sodium hydroxide using a digital pH meter (Fischer Scientific,

NY, USA). pH was also determined using the same pH meter. Firmness

was measured using Instron Universal Testing Machine (model 4502,

Instron Instruments, Massachussetts, USA) set to operate at a crosshead

speed of 25 mm/min, a force of 50 N and using a 5 mm flat steel probe.

The penetration force (N) required to penetrate through the skin was

measured on the equatorial plane by cutting the fruit longitudinally into half

(Batu and Thompson 1998).

The mass, color and visual quality of tomatoes were evaluated for

all the fruits weekly and the destructive tests like TSS, pH and acidity were

determined at the start and at the end of the study period.

Visual quality

The visual quality of the tomatoes was evaluated by subjective

method using a rating scale of 5 points (Aguayo et al., 2004, Ke et al.,

1991 and Talukder et al., 2003). The parameters for evaluation of the

quality of tomatoes are shown below:

Visual Appearance 1 very poor 2 Poor 3 Fair 4 Good 5 Very good

89

Defects* 1 >50%

2 25-50% 3 10-25% 4 10% 5 None

Shrinkage 1 >50% 2 50% 3 25% 4 10% 5 None

*Defects include microbial spoilage, discoloration, pitting and softening

Data analyses

The data was subjected to analysis of variance (ANOVA) and

Duncan’s multiple comparison test of means using the software XL STAT

2006. The results were used to determine the Least Significant

Differences (LSD) amongst treatments at a significance level of 0.05.

4.4 Results and Discussion

The results of the study on the effect of chitosan – lysozyme coatings on

tomatoes stored at optimal conditions of low temperature and high

humidity is discussed below.

90

Mass loss%

Mass loss from the fruits generally occurs as a result of respiration

and transpiration. Edible films are applied over the fruits to reduce this

loss. It is seen from Fig.4.1 that weight loss increased in all the tomatoes

throughout the storage period. The treated tomatoes showed significantly

less mass loss both after 7 days and 14 days compared to the control

(p<0.05).

0

1

2

3

4

5

6

7


Treatment

Mas

s Lo

ss %

7d14d

LSD(0.05)= 0.345

Fig. 4.1: Mean mass loss% of coated and uncoated tomatoes during

storage

Chitosan coating at 2% level was most effective in significantly

(p<0.05) reducing mass loss as seen on both 7 and 14 days. After a

week’s storage there was a greater difference between the treated and

91

untreated samples; this variation was decreased by the second week. On

the whole between the 1% chitosan treated tomatoes no significant

difference was observed. The same was seen even in the case of 2%

chitosan treatment. Lysozyme did not have any influence on the changes

in mass loss. As the relative humidity was high and temperature low, it is

expected to keep the mass loss (due to respiration and transpiration) to

the minimum. These observations are comparable with those observed by

Batu and Thompson (1998) who studied plastic film packaging of pink

tomatoes at 13℃. Tasdelen and Bayindirli (1998) have reported similar

results in their study on Semperfresh coating of tomatoes held at 12℃.

Kittur et al. (1998) and Mathooko (2003) have observed the effect of RH

and temperature on water vapor permeability of edible films.

Color

L* Values

Tab. 4.1: Mean L* values of coated and uncoated tomatoes during storage Means with same letters are not significantly different (p<0.05)

Treatment 0d 7d 14d Control 54.40±0.00 a 48.24±0.00 d 42.15±0.00 d C1 54.16±1.54 a 52.94±0.17 a 48.55±0.29 a C1L0.5 53.66±1.38 a 51.96±0.18 ab 46.51±0.29 bcd C1L1 52.79±1.38 a 50.71±0.18 abcd 47.60±0.29 abcd C1L2 53.85±1.38 a 48.87±0.17 cd 47.83±0.29 abc C2 54.37±1.54 a 50.54±0.18 abcd 48.34±0.29 ab C2L0.5 54.43±1.38 a 51.46±0.17 abc 47.64±0.29 abcd C2L1 53.54±1.38 a 51.00±0.19 abcd 48.75±0.29 cd C2L2 53.17±1.54 a 49.57±0.18 bcd 48.58±0.25 cd

92

Between the coated and uncoated tomatoes there was no

significant difference in the L* values (Tab 4.1). The uncoated tomatoes

showed lower values which indicated that they were darker in color.

Among the treated ones C1, C2, C2L1 and C2L2 were much lighter in

color as shown by their higher L* values. The change in color in these

tomatoes was less. According to Camelo and Gomez (2004) the L* values

indicate the lightness or darkness of the red color of tomatoes during

ripening. They reported that it did not change till the turning stage of

maturity when the green color was still predominant. This value decreased

with ripening as the green turned pink and later to dark red color.

Hue angle

In a ripening tomato, color changes are characterized by

degradation of chlorophyll and accumulation of lycopene (Hobson and

Grierson, 1993).

Tab. 4.2: Mean hue angles of coated and uncoated tomatoes during storage

Means with same letters are not significantly different (p<0.05)

Treatment 0d 7d 14d Control 92.34±0.00 a 66.70±0.00 b 42.83±0.00 c C1 94.16±0.20 a 79.04±0.16 a 55.75±0.30 ab C1L0.5 90.24±0.21 a 78.28±0.17 ab 55.73±0.30 ab C1L1 91.76±0.21 a 78.00±0.17 ab 53.87±0.30 abc C1L2 94.54±0.21 a 74.29±0.16 ab 52.84±0.30 bc C2 92.50±0.20 a 74.94±0.17 ab 59.40±0.30 a C2L0.5 90.48±0.21 a 78.34±0.16 ab 58.53±0.30 abc C2L1 90.67±0.21 a 78.56±0.17 a 57.49±0.30 bc C2L2 91.62±0.20 a 78.39±0.17 ab 58.44±0.27 abc

93

The development of the red color is very essential for the consumer

acceptability of this produce. Tab. 4.2 shows that the coated tomatoes

ripened much slowly than the untreated ones and the changes seem to be

very gradual. At 15℃ the ripening process still took place at a slow rate as

mentioned by Kitinoja and Kader, (1995). At 7 days the hue angles of C1

and C2L1 were found to be significantly different from the control (p<0.05).

Though not statistically significant, the hue angles of the other coated

tomatoes were also much higher than that of the control implying the

effect of coating. By 14 days C1, C1L0.5 and C2 were different (p<0.05)

from the control. No significant difference was seen between 1% and 2%

chitosan treatments, though the color change was slower in 2% chitosan

treatments. C1 appears to show a significant difference from the control

both at 7 and 14 days. Presence of lysozyme did not seem to influence

the ripening process as they do not bring about major changes in the

properties of the film (Park et al., 2004). Pharr and Kattan (1971) observed

that low temperature did not delay the attainment of climacteric maxima

though it reduced rate of respiration and ripening in tomatoes stored at

16℃.

Plastic packaging of tomatoes stored at 13℃ showed that there

was significant difference in color of polyethylene and polypropylene

94

packed tomatoes from unpacked ones indicating the retarding effect of

MAP storage (Batu and Thompson, 1998). At low temperature most of the

metabolic reactions that result in changes in quality are catalysed by

enzymes which are very much dependent on temperature (Kader, 1986).

Appearance

Appearance is an important factor to be considered for good

marketability of any produce. The first visual determinant of quality made

by the buyer is the appearance of fresh fruits and vegetables. Often it is

the most critical factor in the initial purchase besides price, while

subsequent purchases may be more related to texture and flavor

(Mitcham, 1996).

With respect to appearance Fig 4.2 shows that the mean score of

C1 was consistently high until 14 days which was significant at 95%

confidence level. Surprisingly the appearance of untreated tomatoes was

also found to be good until the 7th day, but most of them showed

symptoms of spoilage and dull appearance in 14 days. One per cent

chitosan treatment had a better effect than 2% at 7 days; there was not

much variation seen between the two at 14 days.

The scores for C2, C2L1and C2L2 were low even by 7 days, as

many of them appeared less attractive showing signs of water

95

accumulation and sogginess. This may be due to incompatibility of the

coating at the given conditions of storage. Besides C1, at the end of 14

days C1L0.5 and C1L1 scored better than the untreated tomatoes, though

the scores were not statistically significant. The performance of all the 2%

chitosan films was only fair at 14 days. It is noteworthy that none of the

tomatoes given C1 treatment was spoiled or appeared dull. It appears that

this treatment was good enough to prevent any unfavorable response in

the fruits and also retain the fresh appearance.

0

1

2

3

4

5

6

Control c1 C1L0.5 C1L1 C1L2 C2 C2L0.5 C2L1 C2L2

Treatment

App

eara

nce

(Sco

re)

0d7d14d

LSD(0.05)= 0.688

Fig. 4.2: Mean score for appearance of coated and uncoated tomatoes during storage

Mathooko (2003) reported that after two weeks of storage at 15℃

most of the unwrapped tomatoes had started shriveling, thereby leading to

96

loss of brightness in color and there were signs of mold infection, while

those stored under MAP showed less spoilage.

Disease and defects

Chitosan coatings are reported to prevent spoilage in fruits and

vegetables as it is shown to check the growth of fungi and bacterial

pathogens (Devlieghere et al., 2004; El-Ghouth et al., 1991; Ouattara et

al., 1999). Development of signs of spoilage/disease or defects like

discoloration or blemishes during storage will result in losses. Fig 4.3

shows that on both 7th and 14th day C1 scored maximum (p<0.05) and

was free from defects or spoilage.

0

1

2

3

4

5

6

Control c1 C1L0.5 C1L1 C1L2 C2 C2L0.5 C2L1 C2L2

Treatment

Dis

ease

/Def

ects

(Sco

re)

0d7d14d

LSD(0.05)= 0.675

Fig. 4.3: Mean score for disease and defects in coated and uncoated

tomatoes during storage

97

C1 coating performed very well in maintaining good appearance

and preventing spoilage during the storage period of 14 days. There was

more discoloration, water soaked effect and softening in 2% chitosan

treatments than in 1%. Most of the fruits in these groups showed

symptoms of tissue softening, water accumulation, liquid oozing from the

stem scar and consequently spoilage in 14 days. This could be a sign of

distress or physiological response brought about by the thick 2% chitosan

film. The rupture could have favored the invasion of fungi, humidity being

highly favorable. Addition of lysozyme did not show any additional

protective influence on the quality of the coated tomatoes in this study.

Physiological disorders are adverse quality changes that occur in

fresh produce owing to metabolic disturbances. These disturbances can

be caused by internal factors such as mineral imbalances or may be due

to unfavorable environmental factors such as inappropriate storage

temperatures or atmospheric composition. Mild symptoms are said to be

often confined to superficial tissues which may not be too significant if the

produce is to be processed but can strongly decrease marketability of the

fresh product because of visual disfigurement. Furthermore, physiological

disorders can increase the susceptibility of the commodity to invasion by

pathogens (Paull, 1999; Mathooko, 2003; Guilbert et al., 1996).

98

There was no surface shrinkage as such seen in both treated and

untreated tomatoes due to the high humid conditions of storage.

Firmness

Firmness measurements of coated and uncoated tomatoes (Tab.

4.3) showed that the coated tomatoes were significantly firmer than the

uncoated ones (p<0.05). C1, C1L0.5 and C1L2 were significantly different

Tab. 4.3: Mean firmness (in N) of coated and uncoated tomatoes during stroage

*Initial firmness – 17.51N Means with same letters are not significantly different (P<0.05)

from the other treatments, showing good firm texture. Though the

difference is not very distinct, 1% chitosan treatment gave better firmness

than 2% treatment. This could be attributed to the permeability properties

of the films and its effect on the fruits. Also the lower firmness values in

the 2% chitosan treatments could be due to the softening of tissues

observed in these tomatoes. Application of a film on the tomatoes modifies

Treatment 14d Control 5.37±0.00 bc C1 9.03±0.37 a C1L0.5 8.24±0.37 ab C1L1 7.65±0.37 abc C1L2 8.34±0.37 a C2 7.47±0.37 abc C2L0.5 4.48±0.37 c C2L1 7.33±0.37 abc C2L2 7.05±0.33 abc

99

the atmosphere around the fruit that delays ripening. According to Herner

(1987) softening of fruits can be delayed by reducing respiration through

increasing CO2 concentration.

Batu and Thompson (1998) found that tomatoes exposed to 2-5%

CO2 for 10 days at 12.5℃ developed surface blemishes, excessive

softening and uneven ripening. Firmness also depends on the

wholesomeness of fruits, ie. unfavorable metabolic changes may bring

about loss in turgor. Although some degree of softening is required for

optimal quality in fruit, over softening is undesirable and is a sign of

senescence or internal decay. In some fruits and vegetables (e.g. apples

and tomatoes), the breakdown of intercellular adhesion between cells,

leads to a condition known as mealiness (Aked, 2000).

pH

Normally during the course of ripening pH increases. Fig. 4.4

indicates that pH increased from the initial value of 4.33 in the control and

C2L1 and C2L2 treatments. The control was not significantly different from

the treated tomatoes except in C1. There was a decrease seen in the

other treatments. There was a significantly different (p<0.05) change

observed in C1. No change in pH was seen in C2 treatment. Coating

helps to minimize this change.

100

4.10

4.15

4.20

4.25

4.30

4.35

4.40

4.45

4.50pH


Treatments

14d0d

LSD(0.05)= 0.129

Fig. 4.4: Mean pH of coated and uncoated tomatoes during storage

The low storage temperature also may have played a role in

retarding the biochemical process. The values of pH are well within normal

as given by Cantwell (2000). Tasdelen and Bayindirli (1998) observed

similar fluctuations in pH during low temperature storage of coated

tomatoes.

Titratable acidity

The changes in acid concentration during ripening influences fruit

quality (Batu, 2003). As seen in Fig. 4.5 titratable acidity decreased in all

the tomatoes, both treated and untreated. The untreated tomatoes were

significantly different (p<0.5) from C1, C1L0.5 and C1L1 which showed

lesser changes in acidity. Control level tomatoes had the maximum fall in

101

acidity, besides all the four 2% chitosan treated tomatoes. It appears that

1% chitosan film is able to prevent the decrease in acidity more effectively

0.44

0.46

0.48

0.50

0.52

0.54

0.56

0.58

Treatment

TA %


14d0d

LSD(0.05)= 0.025

Fig. 4.5: Mean titratable acidity of coated and uncoated tomatoes during

storage

than other films. It is not clear whether this decrease in acidity of treated

tomatoes was due to normal senescence or a metabolic response to the

coating. Batu and Thompson (1998) state that modified atmospheric

packaging of tomatoes stored at 13℃ resulted in a fall in acidity. Similar

observations were made by Tasdelen and Bayindirli (1998) in sucrose

polyester coated tomatoes stored at 12℃.

102

Total soluble solids

The major part of the soluble solids in fruits is comprised of sugars

which impart the pleasant taste and flavor on ripening. In this study the

TSS of uncoated tomatoes was very different from the coated tomatoes

though not statistically significant (Fig. 4.6).

3.300

3.400

3.500

3.600

3.700

3.800

3.900

Treatment

TSS

%


14d0d

LSD(0.05)= 0.270

Fig. 4.6: Mean total soluble solids of coated and uncoated tomatoes

during storage

There is increase in some and decrease in the rest. There was a

sharp rise in TSS in the uncoated tomatoes compared to the small

changes seen in C1, C1L1 and C2L0.5. Fall in TSS was more in C1L0.5

and C1L2. It can be observed that the changes, whatever it may be, were

lesser in the treated tomatoes than in the untreated. This implies that

103

ripening did take place in the control at 15℃. The film coating helped to

slow down this change, hence the lower values seen for the treated

tomatoes. The overall change in TSS was small, which could also be due

to low storage temperature. Batu (2003) reported increases in TSS during

tomato ripening at both 15℃ and 13℃, but low O2 storage could also

inhibit sugar accumulation. It is explained that this resulted in suppression

of the metabolism involving conversion of starch to sugars. This decrease

in TSS was seen more clearly at the end of 20 days in film coated

tomatoes stored at 120C (Tasdelen and Bayindirli, 1998).

Shelf life of commodities may differ greatly from laboratory studies

in commercial storage. The distribution chain may not have the facilities to

store each commodity under optimal conditions and therefore

compromises are made on the choice of temperature and relative

humidity. These choices can lead to physiological stress and loss of shelf

life and quality (Paull, 1998). The effects of temperature and humidity

during storage are well known, but it is important to understand its

influence on the sensory attributes of the produce. Chitosan films are

known to modify the internal atmosphere as well as to reduce the

transpiration losses in fruits ( El-Ghaouth et al., 1991). Several studies

104

have been done with chitosan as a coating material ( Zhang and Quantick,

1998; El Ghaouth et al., 1991; Li and Yu, 2001).

This study demonstrates the interactions of low temperature, the

modified atmosphere by film coating and response of the tomatoes. The

chitosan – lysozyme films applied on the tomatoes were effective in

checking moisture loss. Though chitosan films are more permeable to

moisture (Farber et al., 2003), high humidity may have controlled

excessive mass loss.

Of all the films 1% chitosan treatment was most effective in

delaying the changes due to ripening namely hue, firmness, TSS and

acidity. The differences observed between 1% and 2% chitosan

treatments may be due to the increased thickness of film with increase in

concentration. This is expected to change the moisture and gas

permeabilities (Argaiz, 2004). At high humidity, gas permeability also

increases due to greater diffusivity (Farber et al., 2003). There was no

discernable effect of lysozyme on the keeping quality of the coated

tomatoes, though its antimicrobial activity in chitosan films has been

demonstrated in vitro (Park et al., 2004). High humidity was conducive for

microbial spoilage which was noticed in this study. Nevertheless, spoilage

105

was seen only at the stem end or wherever there was a rupture due to

oozing of liquid.

4.5 Conclusion

It is important that the concentration of chitosan be standardized to

suit the produce and its storage conditions. This study points out that 1%

chitosan is sufficient for storage of tomatoes at low temperature and high

humidity.

4.6 References

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110


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111

Chapter V

General Discussion and Conclusion

There is an urgent need to find ways to prevent post harvest losses

in horticultural crops especially in the developing countries, where the

losses are higher. Simple technologies to prevent these losses could be

very beneficial. Application of edible films on a highly perishable

commodity such as tomato to extend its shelf life could bring the farmer

better profits.

Besides checking moisture loss and changes in color and firmness,

1% chitosan-lysozyme films preserved the quality of the tomatoes during

storage. At low temperature high humid conditions the 2% chitosan films

seem to bring about tissue damage and microbial spoilage. Shrinkage was

another problem in this film concentration at low humid conditions. Too

thick films prevent gas and moisture exchange across the film which can

impair the quality of the fruit. C1 was more suitable for optimum storage

and C1L1 was the best for ambient storage. Hence there should be a

prudent selection of chitosan concentration. Plasticizer choice and

concentration may also be modified to change film properties.

Chitosan films have good physical and mechanical properties that

can be very appropriate for fruit and vegetable preservation. Miranda et al.

112

(2004) state that chitosan films were more resistant to mass transfer with

the increase in thickness, thus increasing the water vapor pressure within

the film. Water transport mechanism is said to be more complex as water

sorption isotherms are nonlinear and diffusivity is moisture dependant.

Plasticizers are said to play an important role in film thickness.

Gontard et al. (1996) state that at low RH wheat gluten films

showed very low O2 and CO2 permeabilities. It increased exponentially at

RH > 60% due to the plasticizing effect of water molecules. At high RH

chitosan films are more permeable to these gases.

The application of chitosan films on horticultural crops has to be

investigated also in the context of stage of maturity of the produce and the

storage conditions available in different tropical and temperate countries.

With additives these films can be tailor-made to suit specific needs. Since

they are made from biodegradable sea food waste and it is known to have

no toxic effects, it would be highly beneficial to exploit it gainfully. It has

great potential in finding novel uses in food industry considering its

functional properties.

113

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