Development of Lipid-Based Coatings for the Quality Maintenance of Fruit and Vegetables Doctor of Philosophy Olabisi Abiola Amud
DEVELOPMENT OF LIPID-BASED COATINGS FOR THE QUALITY
MAINTENANCE OF FRUIT AND VEGETABLES
A Thesis
submitted to the
• University of New South Wales
i as fulfilment of the requirement for the degree of
DOCTOR OF PHILOSOPHY
by
OLABISI ABIOLA AMUDIPE
B.Tech (Storage Technol.) FUTA, MAppSc (Food Tech.) UNSW
The University of New South Wales
Sydney NSW 2052
Australia June, 1996 U N S W 13 OCT 1998 -ARY DECLARATION
The candidate, Olabisi Abiola Amudipe, hereby declares that none of the work presented in this thesis has been submitted to any other university or institution for higher degree.
O. A. Amudipe
CERTIFICATE OF ORIGINALITY
of Tcom" ,S ^ ^ Wrk and *»< to ,he be ,
toy ow^o7h^ZthTXZ",en' °f,hiS ,hesis is ,he Product of °n s,y,e- a~ o.z
(Signed) ACKNOWLEDGMENT
I wish to express my appreciation and gratitude to my supervisors, Dr J.E. Patton and Dr
C.M.C. Yuen, for their patient supervision, guidance and understanding during the conduct of this study and their constructive criticism during the preparation of this manuscript.
I am also grateful to Mr. K.J. Scott for his valuable advice and assistance during the course of this study. t
I wish to thank Mr P. Chy, Dr M. Forbes-Smith and my other colleagues in the
Postharvest Section for making the conduct of this study enjoyable.
I am grateful to the Commonwealth Government of Australia for the financial support,
The Australian Centre for Industrial and Agricultural Research for the funding of the project and The Federal University of Technology, Akure-Nigeria for granting me a study leave.
To my wonderful family and friends who have always had great faith in me, I owe an immeasurable depth of appreciation.
And above all, I give God all the glory for the great things He has done. TABLE OF CONTENTS
Acknowledgment i
Table of contents ii
List of tables xi
List of figures xv
List of plates xviii
Abstract xx
1 Introduction 1
2 Literature review * 6
2.1 The fruit and vegetable industry 6
2.1.1 Commercial importance of the fruit and vegetable industry 7
2.1.2 Nutritional importance of fruit and vegetables 9
2.2 Postharvest physiology of fruit and vegetables 10
2.2.1 Classification of produce 12
2.2.2 Respiratory metabolism 16
2.2.3 Ethylene biosynthesis and action 20
2.3 Postharvest handling of fruits and vegetables 22
2.4 Postharvest losses of fruit and vegetables 24
2.4.1 Effect of temperature 25
2.4.2 Effect of humidity 27
2.4.3 Mechanical injury 28 2.4.4 Physiological disorders 28
2.4.4.1 Superficial scald 29
2.4.4.2 Core and flesh browning 31
2.4.4.3 Core flush 31
2.5 Atmosphere modification 32
2.5.1 Controlled atmosphere (CA) storage 33
2.5.2 Modified atmosphere (MA) storage 33
2.5.2.1 Polymeric films 34
2.5.2.2 Commercial coatings 35
2.6 Biochemical and physiological effects of modified atmosphere storage
2.6.1 Effect on respiration 36
2.6.2 Effect on ethylene production and action 38
2.6.3 Effect on compositional changes 40
2.6.3.1 Colour 41
2.6.3.1.1 Chlorophyll degradation 41
2.6.3.1.2 Discolouration 42
2.6.3.2 Texture 43
2.6.3.3 Effect on flavour 44
2.6.4 Effect on physiological disorders 44
2.6.5 Effect on growth and development 46
2.6.6 Effect on decay microorganisms 47
2.7 Edible coatings 47
iii 2.7.1 Principle of edible coatings 47
2.7.2 Historical background 48
2.8 Modified atmosphere effect of edible coatings on fruit and vegetables 50
2.8.1 Water loss 50
2.8.2 Appearance 52
2.8.3 Ripening and quality maintenance 52
2.8.4 Physiological disorders 53
2.8.5 Decay microorganisms 54
2.8.6 Gaseous exchange and/espiration rate 55
2.8.7 Detrimental effect of edible coatings 58
2.9 Development of lipid based edible coatings 59
2.10 Chemistry and properties of lipid coatings 61
2.10.1 Fats and oils 61
2.10.1.1 Chemical properties 62
2.10.1.2 Physical properties 63
2.10.1.2.1 Melting point 63
2.10.1.2.2 Oiliness and viscosity 63
2.10.1.2.3 Surface and interfacial tension 64
2.10.1.2.4 Solubility in water 64
2.10.1.3 Classification, sources and properties 65
2.10.2 Emulsion and emulsifiers 67
2.10.2.1 Definitions 67 2.10.2.2 Types of emulsion 67
2.10.2.3 Emulsifiers 68
2.10.2.4 Types of emulsifiers 69
2.10.2.4.1 Oil and fat derivatives 69
2.10.2.4.2 Hydrocolloids 70
2.10.2.5 Properties and functions of emulsifiers 72
2.10.2.6 Formation of emulsions 73
2.10.2.7 Application of emulsion (wettability) 74 i 2.10.2.8 Wetting of solids 75
2.10.2.9 Nature of wax film 75
2.10.2.10 Thickness of wax film 77
3 Materials and methods 78
3.1 Produce 78
3.2 Coating materials 80
3.3 Preparation and formulation of coatings 81
3.4 Application of coatings 86
3.4.1 Lipid coatings 86
3.4.2 Emulsion 86
3.5 Produce assessment 88
3.5.1 Physico-chemical Analysis 88
3.5.1.1 Weight loss 88
3.5.1.2 Colour assessment 88 3.5.1.3 Measurement of firmness 89
3.5.1.4 Measurement of pH 89
3.5.1.5 Titratable acidity 90
3.5.1.6 Total soluble solids 90
3.5.1.7 Sensory evaluation 90
3.5.1.8 Assessment of physiological disorders 91
3.6 Extraction and analysis of a-farnesene 92
3.7 Extraction and analysis of putrescine 93
3.8 Physiological Measurements ' 95 i
3.8.1 Respiration rate 95
3.8.2 Internal atmosphere 95
3.8.3 Measurement of carbon dioxide and oxygen 98
3.8.4 Measurement of ethylene 98
3.9 Determination of skin permeability 99
3.10 Electron microscopy of coatings 100
3.11 Statistical analysis 100
4 Results 102
4.1 Preliminary studies 102
4.1.1 Non climacteric produce 102
4.1.1.1 Capsicum 102
4.1.1.2 Cucumber 106
4.1.1.3 Carrot 109
VI 4.1.1.4 Zucchini 111
4.1.2 Climacteric produce 114
4.1.2.1 Avocado 114
4.1.2.2 Custard apple 114
4.1.2.3 Mango 115
4.1.3 Summary 117
4.2 Effect of lipid coatings on selected produce 118
4.2.1 Introduction 118
4.2.2 Result , 118
4.2.2.1 Lychee 118
4.2.2.2 Plum 121
4.2.2.3 Nectarine 126
4.2.2.4 Banana 132
4.2.2.5 Apple 138
4.2.2.6 Nashi 147
4.2.2.7 Pear 156
4.3 Formulation trials 166
4.4 Effect of oils and oil emulsions in extending the storage life of produce
4.4.1 Introduction 169
4.4.2 Result 169
4.4.2.1 Cucumber 169
4.4.2.2 Avocado 174
vii 4.4.2.3 Nashi 177
4.4.2.4 Apple 184
4.4.3 Summary 188
4.5 Coating morphology 189
4.5.1 Introduction 189
4.5.2 Result 190
4.5.3 Summary 192
4.6 Effect of oils and oil-based coatings on the permeability of fruit surface to
gases and internal composition of Nashi and apple 192
4.6.1 Introduction 192
4.6.2 Result 190
4.6.2.1 Nashi 193
4.6.2.2 Apple 195
4.6.3 Summary 197
4.7 Effect of oils and oil-based emulsions on respiration and internal gas
composition of coated fruits 198
4.7.1 Introduction 198
4.7.2 Result 198
4.7.2.1 Avocado 198
4.7.2.2 Nashi 200
4.7.2.3 Apple 204
4.7.3 Summary 208
viii 4.8 Effect of coating on physiological disorders and internal atmosphere of
Nashi and apples stored at 0°C 209
4.8.1 Introduction 209
4.8.2 Result 209
4.8.2.1 Nashi 209
4.8.2.2 Apple 215
4.9 Effect of coatings on a-farnesene and putrescine accumulation in Granny
Smith apple during storage at 0°C 221
4.9.1 Introduction , 221
4.9.2 Result 221
4.9.3 Summary 223
5 Discussion 224
5.1 Introduction 224
5.2 Effect of coating on storage life 225
5.3 Mechanism of action of coatings 228
5.4 Effect of coatings on permeability of fruit surface to gases 229
5.5 Effect of coatings on sensitivity of fruit to ethylene 231
5.6 Effect of coatings on respiratory metabolism 232
5.7 Effect of coatings on development of physiological disorder 234
6 Conclusion 239
6.1 Commercial potential of coating 240
6.2 Commercial application of coating 240
ix 6.3 Potential problems 242
6.4 Justification 242
6.5 Recommendation 243
Bibliography 245
X LIST OF TABLES
Table 1. Percentage of vitamin and mineral intake in the Australian diet
contributed by fruit and vegetables (1985 - 1991).
Table 2. Classification of some fruits and vegeatbles.
Table 3. Production and loss figures in less-developed countries for selected
produce.
Table 4. Classification of fruit and vegetables according to their tolerance to low
oxygen concentrations.
Table 5. Classification of fruit and vegetables according to their tolerance to
elevated carbon dioxide concentrations.
Table 6. Surface tension of some oils at different temperatures.
Table 7. Classification, sources and properties of some commercially important
lipids.
Table 8. Some functions of gums in food products.
Table 9. Classification of major hydrocolloids used in food products.
Table 10. Physiological and biochemical properties of produce studied.
Table 11. Brand names and sources of supply of coating materials used.
Table 12. Lipids trialed in preliminary study.
Table 13. Quality parameters assessed after coating produce with saturated,
monounsaturated, polyunsaturated lipids and mineral oil.
Table 14. Evaluation of emulsion stability.
xi Table 15. Quality parameters assessed for the effect of oil and oil-based coatings
on selected produce.
Table 16. Effect of lipid coatings on green colour retention of capsicum at 20°C.
Table 17. Effect of lipid coatings on shrivelling of capsicum at 20°C.
Table 18. Effect of coatings on green colour retention of cucumbers stored at
20°C.
Table 19. Effect of coatings on storage life of carrots stored at 20° and 7°C.
Table 20. Effect of coatings on storage life of zucchini stored at 20° and 7°C.
Table 21. Effect of lipid coatings on the w'eight loss and storage life of lychees at
20°C.
Table 22. Effect of lipid coatings on the physical and chemical characteristics of
Santa Rosa plums after 9 days of storage at 20°C.
Table 23. Effect of lipid coatings on the physical and chemical characteristics of
Santa Rosa plums after 31 days of storage at 0°C.
Table 24. Effect of lipid coatings on firmness and chemical characteristics of
nectarines after 15 days of storage at 20°C.
Table 25. Effect of lipid coatings on firmness and chemical characteristics of
nectarines after 29 days of storage at 20°C.
Table 26. Effect of lipid coatings on firmness and chemical characteristics of
Granny Smith apples after 53 days of storage at 20°C.
Table 27. Shrivelling and rotting of Granny Smith apples after 81 days of storage
Xll at 20°C
Table 28. Effect of lipid coatings on physical and physiological changes of Nashi
after 24 weeks of storage at 0°C.
Table 29. Effect of lipid coatings on changes in chemical compositions of Nashi
after 15 days of storage at 20°C.
Table 30. Effect of lipid coatings on firmness and chemical compositions of pears
after 15 days of storage at 20°C.
Table 31. Effect of lipid coatings on physical and physiological changes of pears
after 30 weeks of storage at 20°C.
Table 32. Formulation trials on selected produce.
Table 33. Effect of oils and oil-based emulsions on the storage life of cucumbers
at 20°C.
Table 34. Effect of oil emulsions on the storage life of Hass avocados stored at
20°C.
Table 35. Sensory attributes of Nashi after 33 days storage at 20°C.
Table 36. Effect of oils and oil-based emulsions on quality attributes and chemical
compositions of Granny Smith apples after 7 weeks storage at 20°C.
Table 37. Sensory attributes of apples after 20 weeks storage at 0°C and 7 days at
20°C.
Table 38. Effect of oil and oil-based coatings on the skin permeability to carbon
dioxide and oxygen and the internal gas composition of Nashi at 20°C.
xiii Table 39. Effect of oil and oil-based coatings on the skin permeability to carbon
dioxide and oxygen and the internal gas composition of Granny Smith at
20°C.
Table 40. Effect of oils and oil-based coatings on the development of
physiological disorders in Nashi after 11 months storage at 0°C.
xiv LIST OF FIGURES
Figure 1. Apparent consumption of fresh fruit and fruit products per person per
year during the past 50 years in Australia (ABS, 1990).
Figure 2. Percentage distribution of weekly average household expenditure on
(a) total food and non alcoholic beverages (b) fruit and (c) vegetables in
Australia for 1988 - 1989 (ABS, 1989).
Figure 3. Growth patterns of fruit during development.
Figure 4. Utilisation of substrate and oxygen and the formation of carbon dioxide,
water and energy during respiration.
Figure 5. Pathway of glycolysis.
Figure 6. Reactions of Krebs cycle.
Figure 7. Schematic diagram of handling of fruit and vegetables from harvest
through to consumer.
Figure 8. Effect of lipid coatings on weight loss of nectarines stored at 20°C.
Figure 9. Effect of lipid coatings on weight loss of nectarines after 29 days of
storage at 0°C.
Figure 10. Effect of lipid coatings on colour changes of bananas stored at 20°C.
Figure 11. Effect of lipid coatings on weight loss of bananas stored at 20°C.
Figure 12. Effect of lipid coatings on (a) firmness and (b) weight loss of Granny
Smith apples stored at 20°C. Figure 13. Effect of lipid coatings on (a) colour, (b) firmness and (c) weight loss of
Nashi stored at 20°C.
Figure 14. Effect of lipid coatings on colour changes of pears stored at 20°C.
Figure 15. Effect of lipid coatings on weight loss of pears stored at 20°C.
Figure 16. Effect of oils and oil-based emulsions on colour, shrivelling and weight
loss of cucumber stored at 20°C.
Figure 17. Effect of oil emulsions on weight loss, colour and firmness of avocado
stored at 20°C.
Figure 18. Effect of oils and oil-based emtilsions on colour changes and weight loss
in Nashi stored at 20°C.
Figure 19. Changes in firmness, total soluble solids, titratable acidity and pH of
Nashi at 20°C.
Figure 20. Effect of coating on respiration rate and internal gas composition of
avocados stored at 20°C.
Figure 21. Respiration rate of Nashi stored at 20°C.
Figure 22. Effect of oils and oil-based emulsions on the internal oxygen, carbon
dioxide and ethylene composition of Nashi at 20°C.
Figure 23. Respiration rate of Granny Smith apples at 20°C.
Figure 24. Effect of oils and oil-based emulsions on the internal oxygen, carbon
dioxide and ethylene composition of Granny Smith apple at 20°C.
XVI Figure 25. Effect of oils and oil-based emulsions on the internal oxygen, carbon
dioxide and ethylene composition of Nashi during storage at 0°C and
subsequent holding at 20°C.
Figure 26. Effect of oils and oil-based emulsions on the internal oxygen, carbon
dioxide and ethylene composition of Granny Smith apples at 0°C and
subsequent holding at 20°C.
Figure 27. Changes in a-farnesene and putrescine levels of Granny Smith apples
stored at 0°C.
XVII LIST OF PLATES
Plate 1. Apparatus used for vacuum extraction of the internal gases.
Plate 2. Coated bananas after 18 days storage at 20°C.
Plate 3. Effect of lipid coatings on chilling injury of bananas after 3 days
storage at 7°C a) + 1 week and b) + 2 weeks at 20°C.
Plate 4. Coated Granny Smith apples after a) 7 weeks and 13 weeks at 20°C
Plate 5. Granny Smith apples coated with different oils after 17 weeks storage
at 0°C + 1 week at 20°C. ,
Plate 6. Nashi coated with different lipids after 19 days of storage at 20°C.
Plate 7. Nashi coated with different lipids after 28 weeks of storage at 0°C + 1
week at 0°C.
Plate 8. Pears coated with different lipids after 22 days of storage at 20°C.
Plate 9. Pears coated with different lipids after 30 weeks at 0°C + 12 days at
20°C.
Plate 10. Coated cucumbers after 28 days of storage at 20°C.
Plate 11. Nashi coated with oils and oil-based emulsions after 26 days storage at
20°C.
Plate 12. Electron micrographs of the surface of uncoated apples (A) and those
coated with sunflower oil (B), 3.33% (w/w) sunflower emulsion (C)
and 3% sweet potato starch (D) after (i) 0; (ii) 4 and (iii) 7 weeks
xviii of storage at 20°C.
Plate 13. Effect of oils and oil-based emulsions on a) superficial scald and b)
core and flesh browning of Nashi after 47 weeks storage at 0°C + 9
days at 20°C.
Plate 14. Effect of oils and oil-based coatings on superficial scald of apples after
20 weeks storage at 0°C a) + 1 week and b) 3 weeks at 20°C. ABSTRACT
A range of lipid and lipid based skin coatings were developed and evaluated for their effectiveness in maintaining the quality of selected fruits and vegetables under ambient and low temperature storage. Most coatings tested, irrespective of the lipids’ chain length or degree of saturation were promising in extending the storage life of Nashi, apple, lychee, plum, nectarine, cucumber, carrot and capsicum. The magnitude of shelf life extension varied between produce and the type of coating used.
4
During storage at 20°C, the coatings significantly delayed the development of browning in lychees by 4 - 7 days, shrivelling in carrots and capsicums by 9 and 18 days respectively, and yellowing in cucumber by 30 days. Ripening in Nashi and apple was markedly delayed by 16 and 35 days, respectively. Normal ripening was adversely affected when the lipid coatings were applied at too high concentrations, with mango, custard apple, avocado and pear failing to ripen properly.
A composite coating consisting of 10% sunflower oil, 1% tragacanth gum, 1% Agral and 88% water and applied as a dip, was effective in delaying ripening of avocado, apple and Nashi, by retarding softening in the fruit, and yellowing in Granny Smith apples Nashi and pears. Coatings appear to exert their effect on quality and storage life by modifying the diffusion movement and accumulation of oxygen, carbon dioxide and ethylene through the skin of the produce and within the produce, respectively. The permeability of the apple and Nashi skins to oxygen and carbon dioxide was drastically reduced, resulting in a depressed oxygen level and accumulation of carbon dioxide within the fruit tissue.
This resulted in delayed ripening and reduced respiratory activity, as well as reduced sensitivity of the fruit tissue to ethylene and chilling injury.
Lipid coatings significantly reduced the incidence of superficial scald in Granny Smith i apple and senescence scald, core and flesh browning of Nashi at 0°C. Coatings were found to reduce the production of a-farnesene and also retarded the accumulation of putrescine.
The oil emulsion developed in this work was shown to have potential as a commercial treatment for quality maintenance of fruits and vegetables, and chilling injury resistance.
XXI 1 INTRODUCTION
Fruit and vegetables are considered important components of the human diet as they are the main source of dietary ascorbic acid, as well as being high in dietary fibre. They also contain significant amounts of carbohydrate and minerals and are of considerable culinary significance. The annual per capital consumption, especially for fresh fruit, has increased steadily during the last four decades (ABS, 1990).
Fresh produce are highly perishable and rapidly lose their quality after harvest. About a quarter of the world's fruit and vegetables perish each year prior to reaching the
r consumer and in some tropical countries entire crops are lost between farm and market.
In addition to serious loss of produce quantity, quality, nutrition, value and consumer acceptability, the lack of appropriate shelf-life extension technology in some areas also means that fruit and vegetables produced within a production region can only be consumed by the population within the immediate vicinity, limiting distribution and export markets.
Techniques used to preserve fresh fruits and vegetables during transportation, storage and distribution are generally based on refrigeration with or without control of ambient gases. The overall objective is to slow down respiration and disease development in order to extend produce life. Refrigerated storage is not beneficial to chilling sensitive produce and long term storage at low temperatures results in the development of physiological disorders such as superficial scald on apples, pears and Nashi.
1 Control of ambient atmosphere has been achieved through Controlled Atmosphere (CA) and Modified Atmosphere (MA) storage techniques. CA storage, which involves the removal and/or addition of different gases into the storage room, has been extensively used in conjunction with refrigeration to extend the storage life of apples and has been recommended for other fruits and vegetables (Geeson and Browne, 1980, Geeson,
1984). Economically, these storage units are costly to construct and maintain, as well as having limited availability in many countries.
Modified atmosphere on the other hand is achieved by use of non-edible films or edible
r commercial coatings.
The most commonly used non edible films in the postharvest handling of fresh horticultural produce are the polymeric films. The films are utilised in the form of liners, bags or individual wraps. Although films are relatively cheap and simple to employ, the use of polymeric films on produce could be considered as high technology in some developing countries given the fact that particular films with the appropriate characteristics ie permeability, thickness, must be obtained and the ratio of surface area to enclosed produce must be maintained (Lowings and Cutts, 1982), which may require specialised packaging equipment.
Several types of edible commercial coatings have been developed to preserve quality and extend the postharvest life of fruits and vegetables. The most notable “coating material”
2 is a mixture of sucrose esters of fatty acids and the sodium salt of carboxyl methycellulose which is marketed under the trade names of ‘Tal-Prolong’ and
‘Semperfresh’. These coatings were originally developed with a view to providing a cheaper, simple to apply, more ecologically and aesthetically acceptable alternative to non-edible films and coatings and expensive controlled atmosphere storage facilities
(Lowings and Cutts, 1982). Unfortunately, often contrary to product claims, numerous scientific assessment studies on ‘Prolong’ and ‘Semperfresh’ have shown that these coatings have only limited postharvest life enhancing properties on fresh produce
(Peacock, 1988; Yuen, unpublished data). The coatings are not cheap (‘Semperfresh’,
f A$ 150/kg) and are known to be time-consuming and cumbersome to prepare for application. ‘Prolong’ and ‘Semperfresh’ coated-products are also known to frequently behave in an unpredictable and unreliable manner when handled under commercial, rather than laboratory conditions.
Presently, all currently available edible and non-edible commercial coatings such as waxes, ‘Prolong’, ‘Semperfresh’ and polymeric films are difficult to obtain and too expensive for the average farmer in developing countries. Most of these are not manufactured locally or imported in sufficient quantity to meet demand. Governmental regulations in some countries also do not permit direct importation of these products by producers. It is however vitally important that the materials the handlers need to improve postharvest handling are readily available.
3 The successful development and implementation of coating technology involving use of commonly available food ingredients such as lipids and starches is likely to have considerable benefits for Australia as well as in developing countries.
Apple, pear and Nashi are of major importance in Australia in terms of domestic consumption, economic value, and export. Many varieties of these fruits are however
susceptible to the physiological disorder superficial scald. There is an increasing
international pressure to ban or reduce the chemical agent (DPA) currently being used to
control this disorder, creating an urgent need to develop alternative control measures.
r An edible coating which can reduce or possibly completely eliminate scald problem will
be of potential economical benefit to farmers in general. It is also conceivable that the
successful application of edible coatings to some tropical fruits could result in the
development of new export markets for some products where previously distance and
cost of air or refrigerated transportation were prohibitive.
The aim of this study is to develop modified atmosphere packaging in the form of lipid-
based edible coatings which are relatively simple to apply, safe and cost-effective, but at
the same time, easily accessible and affordable to the average farmer in developing
countries. The coating should be suitable for quality maintenance and shelf life extension
of commercially important fruits and vegetables under ambient and low temperature
conditions.
4 The objectives of this study are: i) to assess lipid materials for their coating properties ii) to formulate simple edible coatings from lipid materials and other food ingredients
such as gums and starches
iii) assess the ability of the formulated coatings to extend quality and storage life of a
variety of fruit and vegetables
iv) to determine if the coatings act as a form of modified atmosphere packaging by
measuring respiration rates, internal atmospheres, and alteration to skin surface
permeability
f v) to assess if the coatings can increase resistance of chilling sensitive produce to low
temperatures.
This is to be achieved by systematically testing and developing a number of edible
coatings based on simple food ingredients such as fats, oils gums and starches. The
performance of the different coatings on weight loss, firmness, chemical composition and
respiration rate of some fruits and vegetables are examined. The mode of action of the
coatings is also established.
5 2 LITERATURE REVIEW
2.1 THE FRUIT AND VEGETABLE INDUSTRY
Consumer demand for fresh, low-calorie, healthy, nutritious, high quality foods has increased markedly over the past 10 years (Powrie and Skura, 1991). Fruit and vegetables are now of significant dietary importance due to their low calorific value, low fat and cholesterol, and high fibre properties. They are also the main source of dietary ascorbic acid (vitamin C).
Fruit and vegetable consumption patterns vary with consumer attitudes, demographic changes, availability of processing technology, distribution alternatives, cost of raw material production and environmental issues (Dougherty, 1990). According to a 1990
Horticulture Research and Development Cooperation (HRDC) study, Australians have become increasingly conscious about health and nutrition and are concerned with the freshness of food consumed.
There has been a steady increase in fresh fruit consumption in Australia compared to preserved, dried and processed fruit (Figure 1) (ABS, 1990). According to the 1988 -
1989 household expenditure survey, the average Australian household spent 14% of total food and non alcoholic beverage expenditure on fruit and vegetables. Eighty four percent of the money spent on fruit was on fresh fruit. Similarly, 79% of the total vegetable expenditure was on fresh vegetables (Figure 2) (ABS, 1989).
Fruit and vegetables are therefore of both commercial and nutritional importance as evidenced by increased levels of demand. Postharvest handling practises must be able to ensure continued supply of high quality fruits and vegetables.
6 90
80
88-39 48-49 58-59 68-69 78-79 88-89 90-91 t Time
Figure 1. Apparent consumption of fresh fruit and fruit products per person per year during the past 50 years in Australia (ABS, 1990)
2.1.1 Commercial importance of the fruit and vegetable industry
As an agricultural industry, the Australian fruit and vegetable sector is important in terms
of value, investment and the number of people employed in the production, handling,
transportation and marketing of the commodities. In Australia, there is a fast growing
-export trade in fresh produce which is presently estimated at $250 million a year (Yuen,
1991). Fruit and vegetables, including nuts and grapes, comprised 38% of the gross
value of edible crops produced in Australia during 1992 - 1993, and made up 14% of
the total value of all agricultural commodities (ABS, 1993).
7 A - Total food end non clchciic beverages
fruit tv vegetables
E6V
others
B - Fruit f C - Vegetables
dried processed processed 11V
fresh fresh
Figure 2. Percentage distribution of weekly average household expenditure onn
(a) total food and non alcoholic beverages (b) fruit and (c) vegetables in
Australia for 1988 -1989 (ABS, 1989)
8 2.1.2 Nutritional importance of fruit and vegetables
Fruit and vegetables provide aesthetic appeal, taste, variety and culinary enjoyment. As well as being the main source of vitamin C, they supply substantial amounts of vitamin A, thiamine, valuable amounts of niacin, riboflavin and minerals such as potassium, iron and calcium (Powrie and Skura, 1991). In the Australian diet, 90% of vitamin C, 20% of thiamine and 20% of iron are supplied by fruit and vegetables (Table 1).
Next to water, carbohydrates are the most abundant nutrients supplied by fruit, present in the form of low molecular weight sugars and high molecular weight polymers such as
starch, hemicellulose, cellulose and pectic substances. Hemicellulose, cellulose, pectic
substances and lignin constitute dietary fibre. Their presence along with organic acids
give fresh fruit a natural laxative effect.
Table 1. Percentage of vitamin and mineral intake in the Australian diet contributed by fruit and vegetables (1985 - 1991)
Nutrient Fruit Vegetables Total
Calcium 4.5 ± 0.24 4.9 ± 0.23 - 9.4 Iron 6.5 ± 0.36 15.5 ±0.55 22.0 Vitamin A 1.5 ± 0.13 18.0 ±1.90 19.5 Ascorbic acid 39.0 ±0.86 51.0 ±2.00 90.0 Thiamine 7.0 ±0.58 13.8 ±0.52 20.8 Riboflavin 2.8 ±0.11 6.6 ± 0.41 9.4 Niacin 2.5 ± 0.08 13.5 ±0.35 16.0
Calculated using Australian Bureau of Statistics data on Level of Nutrient Intake
9 2.2 Postharvest physiology of fruit and vegetables
Physiological development of fruit and vegetables is basically divided into three major stages following initiation of germination (Biale, 1964). These are growth, maturation and senescence (Figure 3).
The growth phase is characterised by cell division and cell enlargement and determines the final size of the fruit. The latter part of the growth phase coincides with the initial stage of maturity. Ripening of fruit occurs during the latter part of maturity and the initial stage of senescence. It is an irreversible process in climacteric fruit, converting a physiologically mature inedible fruit to an edible and visually attractive state.
The major changes during ripening are an' increase in respiration rate, ethylene production and membrane permeability, softening of tissues, changes in carbohydrate composition, acidity and protein, production of volatile and pigments and development of waxes on the fruit surface (Biale, 1964; Pratt and Goeschl, 1969).
Senescence, a period when anabolic (synthetic) biochemical processes give way to catabolic (degradative) processes is of great concern in postharvest practices (Wills et al., 1989). Senescence causes the weakening of cellular structure and membrane integrity, which is a result of lipid breakdown, disorganisation of glycolipids and the production of free acids which are toxic to cellular processes (Mazliak, 1983). This leads to aging and finally death of the tissue. This stage, although not preventable, can be delayed.
Fruits are harvested either after the completion of the development and maturation stages or when mature or ripe depending on the end use. Harvesting results in the cessation of the food and water supply to the fruit from the parent plant. Harvested fruit
10 remain alive and therefore use up their reserved food supply in order to provide the energy needed for their metabolic and physiological activities.
The respiration process is the sole source of this energy and results from the breakdown of the plant food reserve - mainly the carbohydrate substrate - along with the production of carbon dioxide and water (Figure 4).
Fruit growth
Respiration climacteric
Maturation
Cell enlargement
Figure 3. Growth patterns of fruit during development. (Adapted from Biale
(1964))
11 Figure 4. Utilisation of substrate and oxygen and the formation of carbon dioxide,
water and energy during respiration ( Kays 1991).
2.2.1 Classification of produce
Fruits are classified as climacteric or non-climacteric based on respiratory patterns during ripening.
Climacteric fruits are those in which ripening is associated with a distinct increase in respiration. Non-climacteric produce on the other hand, exhibit a simple gradual decline in respiration throughout maturation and senescence (Figure 3).
All vegetables (except some fruit-vegetables, such as tomatoes) are considered to have a non-climacteric type of respiratory pattern (Wills et al., 1989).
12 Another distinguishing feature of climacteric and non-climacteric produce is changes in ethylene production during ripening (McGlasson, 1985). All fruits produce minute quantities of ethylene during development. Coinciding with ripening, climacteric fruits produce a larger increase in ethylene while non-climacteric fruits change little throughout development and ripening (Biale and Young, 1981).
Response to applied ethylene by climacteric fruits is noted only during the preclimacteric phase, whereas in non-climacteric fruits respiratory activity and ripening can be accelerated at all stages of maturity (Salunkhe and Wu, 1976). Table 2 shows the classification of some fruits and their internal ethylene concentration.
Fruits and vegetables can be further classified 6ased on their perishability, sensitivity to chilling temperature, respiration rate and internal ethylene concentration. Table 2 gives a summary for the classification of some produce.
13 Table 2. Classification of some fruits and vegetables
Perishability Very perishable Perishable Non-perishable Banana Avocado Apple Mango Nectarine Pear Cucumber Plum Nashi Zucchini Lychee Custard apple
Respiratory pattern Climacteric Non-climacteric Apple Cucumber
Avocado Nashi r Banana Capsicum Custard apple Zucchini Mango Pear Plum
Respiration rates High Medium Low Zucchini Avocado Apple Mango Banana Capsicum Lychee Nectarine Cucumber Plum Nashi Pear
Sensitivity to chilling temperature High (tropical) Medium (sub-tropical) Low (temperate) Banana Avocado Apple Mango Capsicum Nashi Cucumber Pear Lychee Nectarine Zucchini Plum
14 Table 2 (contd) Internal ethylene concentration
High Medium Low Apple Avocado Banana Pear Mango Nectarine Plum Nashi Capsicum Cucumber Zucchini Biale and Young (1974), Wills et al., (1989)
15 2.2.2 Respiratory metabolism
The most important process in fresh produce is respiration which is essentially a biochemical oxidation typical to all living cells. Respiration is a good index of general metabolic activity, indicating the rate of oxidation of respiratory substrate and final deterioration. Respiration rate is therefore, roughly related to the potential storage life of a commodity.
It is generally considered that a slower rate of utilisation of carbohydrates, organic acids, and other reserves leads to prolonging the life of produce (Wills et al., 1989).
Respiration in the cells of fruits and vegetables is the metabolic process involving the breakdown (ie catabolism) of complex organic compounds such as sugars, organic acids, amino acids and fatty acids into lower molecular weight molecules. This in turn produces energy (ATP and heat) through oxidation and reduction enzymic reactions.
Adenosine triphosphate (ATP) carries the free energy needed to (1) sustain metabolic reactions in cells; (2) transport metabolites and (3) maintain cellular organisation and membrane permeability (Powrie and Skura, 1991).
The development of full-bodied flavours and deep colours of harvested fruits is dependent on anabolism for the synthesis of odoriferous compounds and pigments
(which is part of the ripening process). The metabolic precursors and the high-energy nicotinamide adenine dinucleotide phosphate H (NADPH) needed in these synthetic reactions may be derived from the pentose phosphate pathway of the respiration process.
Aerobic respiration includes stepwise energy-releasing reactions in which organic compounds are oxidised to CO2 and the absorbed O2 is reduced to H2O. This is simply represented as:
16 C2H12O6 + 6O2 6CO2 + 6H2O + energy (ATP)
It involves 4 pathways: glycolysis, pentose phosphate pathway, Krebs or citric acid cycle and oxidative phosphorylation. Respiration under anaerobic conditions is however limited to the glycolysis and pentose phosphate pathway systems.
Glycolysis takes place in the cytosol of plant cells and involves enzymic breakdown of glucose or fructose to pyruvic acid with no oxygen entering the pathway or CO2 being released (Powrie and Skura, 1991). The glycolysis pathway for glucose is presented in
Figure 5.
Under sufficient oxygen atmosphere, pyruvate which has migrated into the mitochondrial matrix from the cytosol is oxidatively decarboxylated to an acetyl unit which combines with coenzyme A (Co A) to form acetyl Co A. The acetyl unit enters into the Kreb cycle with two carbons which later leave as two CO2 molecules (Figure 6).
Under low oxygen conditions, the glycolytic pathway replaces the Krebs cycle as the main source of the energy needed for the plant tissues. Pyruvic acid instead of being oxidised to an acetyl unit is now decarboxylated to acetaldehyde with the formation of
CO2 and ultimately ethanol, which results in development of off-flavours and tissue breakdown.
Prolonged exposure of plant tissues to anaerobic conditions leads to death of cells (Kays,
1991). Although the O2 level at which anaerobic respiration occurs could be as low as
0.2% within the plant cell; the gradient of O2 concentration from the cell to the external atmosphere requires about 1-3% O2 around the commodity for maintenance, and this depends on the respiration rate and gas diffusion characteristics of the dermal and subdermal tissues of the specific cultivar of the given commodity (Burton, 1978).
17 GIuoom I AyP
|*ADP
GIocom 6-phosphate
P**o*e*»og»we»«# I
fnictoM 6-phosphate <*««•. 1^ATP utw L pAOP
Fructose 1,$4>itphosp^t0 At&oL*** T
1 Orhydeoxyaoetooer GlycerakJehytJe phosphate 3-phosphate
I^NAO* 4 P,
^ NAOH 4 .H*
U-8(sphosphogfYC 3-Phosphoytycera te r»»oio 2-Phosphoglycerate (noUve ^HjO Phosphoenolpyruva te i^AOP k<«*« \at p Pyruvate Figure 5. Pathway of glycolysis (from Ooraikul and Stiles, 1991) 18 0W°H I C-O ih***rw*« pyt opNosetute. covuyma MAO' C-SCoA CoASH COOH COOH H* ♦ NAOH HO-C-COOH COOH, COOH COOH HOCH m#Ac COOH COOH HCOH HOOC-CH COOH COOH COOH COOH NAO* COOH ATP SCoA COOH COOH *c«d NAO* COOH CoASH SvCC**C *cid ttuokifXMl* toCC*oy< CoA Figure 6. Reactions of Krebs cycle (from Ooraikul and Stiles, 1991) I* 2.2.3 Ethylene biosynthesis and action Ethylene is a plant hormone which regulates many aspects of growth, development, and senescence (Abeles, 1973). It is considered to be aui inducer of the ripening process in many fruits (Lieberman, 1979). Ethylene accelerates loss of green colour which is undesirable in produce such as cabbage, cucumber, lemon and lime. Textural attributes of cucumber were also found to be affected by ethylene concentration (Poenicke et al., 1977). Other undesirable effects of ethylene include bitter taste in carrot (Chalutz et al., 1969, Sarkar and Phan, 1974), and undesirable flavours in sweet potatoes (Buescher et al., 1975). Chilling sensitivity was also reported to increase with ethylene treatment in Haas avocado (Chaplin et al., 1983) and 'Bearss' lemons (McDonald et al., 1985). r McMurchie et al. (1972) proposed that there are itwo systems capable of producing ethylene in plant tissues confirming the suggestion th at both methionine and unsaturated fatty acids may function as substrates for ethylene production in fruits (Lieberman and Mapson, 1964). Methionine has however been considered by many workers to be the sole precursor of ethylene (Rhodes, 1980; Apelbaum et al., 1981). Adams and Yang (1979) explained the sequence for the pathway of ethylene biosynthesis in ripening apples, a pathway that has since been showrn to be operative in all other tested plant tissues. Ethylene is accepted to be producedl from methionine by a pathway involving: (1) the formation of S-adenosyl-methioninte (SAM) in the presence of ATP; (2) the conversion of SAM to 1-aminocyclopropane-1--carboxylic acid (ACC) catalysed by ACC synthase; and (3) ACC oxidation (oxygen required) and deamination to ethylene. 20 Oxygen may be required for ethylene synthesis at two sites. The first being the requirement for ATP generated in respiration for the activation of methionine (ie the recycling of 5-methylthioadenosine to methionine) and the second, the breakdown of ACC formed by the cleavage of S-adenosyl methionine to ethylene (Adams and Yang, 1979; Wang, 1990). Knee (1980) suggested that the second stage is more dependent upon oxygen as he observed an accumulation of ACC under nitrogen. In other words, there was still production of ACC. The inhibition of ethylene synthesis in low oxygen storage according to Knee (1980) is therefore the later stage ie the breakdown of ACC to ethylene. Similar results of accumulated ACC and reduced ethylene production under anaerobic conditions were also reported by Yang (1985) and Banks et al. (1985). Burg and Burg (1967) observed that the structural requirements for biological activity were similar to the stability constants of olefin-silver complexes, leading them to propose the reversible binding of ethylene to a metal-containing receptor site in order to produce a biological effect. Trewavas (1982) summarised the relationship of a plant growth substance with its host tissue in the equation : Growth substance (GS) + receptor (R) (GS.R) biological response. Ethylene is therefore thought to bind to a receptor, forming an activated complex which in turn triggers the primary reaction. The primary reaction then initiates the chain reactions which lead to a wide variety of physiological responses. 21 2.3 POSTHARVEST HANDLING OF FRUIT AND VEGETABLES There has been an increased production of fruit and vegetables worldwide, not only to serve the fresh produce market but also important for the minimal processing and canning industries (Powrie and Skura, 1991). The value of harvested fruit and vegetables increases many times as produce moves from the farm to the retailers through various marketing channels. Therefore, postharvest losses at any point of the distribution chain can cause serious economic loss to everyone involved in the production and marketing of fresh produce. Careful handling and transportation are necessary to reduce postharvest losses. The way in which harvested products are to be utilised determines how they are handled during the postharvest period. Extreme care and effort is required for of produce intended for fresh marketing. Figure 7 summarises the handling stages of fruit and vegetables. Apart from being removed from their source of nutrient supply, in this case the mother plant, fruit and vegetables also have some of their natural protective mechanisms destroyed during postharvest handling. Kaplan (1986) in an overview of washing and waxing of citrus, explained the change that occurs in the wax layer. Citrus fruit secretes a surface layer of natural wax, which contains long-chain alcohols combined with fatty acids, as it matures on the tree. During the heat of the day, the wax softens allowing a heavy residue of dust, dirt, mould, bacterial spores, and preharvest spray residues to adhere to the fruit surface. Although this layer of dirt is often harmless, it does detract from the fruit's appearance and spores carried can infect the fruit and cause decay. It is therefore often necessary in a commercial packing line to wash fruits thoroughly over revolving brushes. Fungicides can also be added, especially if a long storage period is intended. 22 Plant or plant product harvest transport to packing house I Marketing ^— —^ Processing —^ Export 4 Consumer Figure 7. Schematic diagram of handling of fruit/vegetable from harvest through to consumer (How, 1991) 23 Washing the fruits may also remove most of the natural waxes of the cuticle. This results in two undesirable side effects that can limit the market life of the fruit. There is an increase in the rate of water loss and permeability of the cuticle to gases. This results in an unimpeded free entry of oxygen thus increasing the respiration rate of the fruit. The high rate of respiration results in a decreased sugar content, loss in flavour, poorer texture and quality, and a reduction in ascorbic acid. This eventually results in fruit senescence and an increase in decay. These changes occur throughout the remaining stages of the postharvest chain. Appropriate handling techniques ie storage temperatures or postharvest treatments such t as coatings can assist in the control of the rate of these changes. 2.4 POSTHARVEST LOSSES OF FRUIT AND VEGETABLES A postharvest loss is defined as any change in the quantity or quality of a product after harvest that prevents or alters its intended use or decreases its value (Kay, 1991). Postharvest losses vary greatly in kind, magnitude, and where in the postharvest handling system they occur. Serious loss of produce after harvest is a worldwide problem, although it is generally considered to be less in developed countries than developing ones. It is estimated that a quarter of the world's fruit and vegetable supply perish each year before they reach the consumer and entire crops can be lost between farm and market in some tropical countries (Yuen, 1991). Table 3 details losses for some crops. 24 Major factors responsible for postharvest losses of fruit and vegetables include temperature, humidity, mechanical injury, physiological disorders and atmosphere composition. 2.4.1 Effect of temperature Respiration, one of the processes that characterise fruit and vegetables as living entities, involves many enzymatic reactions. These reactions are temperature dependent, with the enzymes remaining active within a physiological temperature range. Temperature is therefore considered a critical factor in the maintenance of quality of fresh produce since it determines the rate of respiration and ultimately the storage life of produce (Snowdon and Ahmed, 1981). 25 Table 3. Production and loss figures in less-developed countries for selected produce Production Estimated loss Commodity (x 1,000 tonnes) (% total crop) Roots/Tubers Carrots (Daucus carota, L.) 557 44 Potatoes (Solarium tuberosum, L.) 26,909 5-40 Sweet potatoes (lpomoea batatas, L Lam.) 17,630 35-95 Yams ([Dioscorea spp.) 20,000 10-60 Cassava (Manihot esculenta, Crantz.) 103,486 10-25 Vegetables Onions {Allium cepa, L.) 6,474 16-35 Tomatoes (,Lycopersicon esculentum, Mill.) 12,755 35-95 Plantain {Musa spp.) 18,301 35-100 Cabbage {Brassica oleracea, L. Capatita) 3,036 37 Cauliflower {Brassica oleracea, L. Botrytis) 916 49 Lettuce {Lactuca sativa, L.) - 62 Fruits Bananas {Musa spp.) 36,898 20-80 Papayas {Carica papaya, L.) 931 40-100 Avocados {Persea americana, Mill.) 1,020 43 Citrus {Citrus spp.) 22,040 20-95 Apples {Malus sylvestris, Mill.) 3,677 14 Adapted from Kays (1991). 26 The activity of enzymes in fruit and vegetables declines at temperatures above 30°C although the temperature at which specific enzymes become inactive varies. For most produce, when held above 35°C, metabolism becomes abnormal and results in a breakdown of membrane integrity and structure, with disruption of cellular organisation and rapid deterioration of the produce (Wills et al., 1989). Refrigeration is the main technique used for reducing storage temperature, subsequently prolonging the shelf life of fruit and vegetables. The lower limit for normal metabolism is the freezing point of the tissues which is usually between 0° and -2°C. Holding produce below this results in freezing which disrupts the interchange of metabolites r between the various cellular components, with the tissue failing to resume normal metabolism or regain normal texture after thawing. Chilling injury and other metabolic (physiological) disorders can set in for most tropical and subtropical fruits which are sensitive to low-temperature storage. Low temperature storage can also alter the starch-sugar balance in some produce such as potato, sweet potato, peas and sweet com, which may or may not be desirable (Wills et al., 1989). 2.4.2 Effect of humidity Humidity affects many aspects of postharvest horticulture, including moisture loss from produce, condensation of moisture on produce, wound healing and survival, growth and pathogenicity of microrganisms. The difference between the humidity in the plant tissue and the humidity in the air of the storage atmosphere controls the moisture loss from tissues. Generally a 5 - 10% loss of water from fruit and vegetables results in loss of turgidity, causing wilting, poor appearance and loss of saleable weight which is a direct 27 loss in marketing (Robinson, et al., 1975). Water loss can also cause a loss of crispness and undesirable changes in colour and palatability of some fruit and vegetables. 2.4.3 Mechanical injury Fruit and vegetables are susceptible to mechanical injury/wound during harvesting, handling and transportation. Other factors such as insects, animals, wind and rain can also inflict injury on fruit and vegetables while in the field. Wounds can be in the form of surface punctures, cuts or lesions with the degree of injury depending on produce type, degree of cell dehydration, stage of maturity, weight and size, skin characteristics of the produce and environmental conditions (Voisy et al., 1970; Hankinson and Rao, 1979; Wilcockson et al., 1980). Mechanical injury results in serious postharvest losses in fruit and vegetables with the actual loss being physical, physiological, pathological or qualitative. Injury increases the respiration rate, ethylene production and water loss from injured tissue (Boehm, 1987). Most importantly, however, is the facilitation of infection by microorganisms leading to visible defects and latent damage to produce (Uritani and Asahi, 1980; Hung, 1993). 2.4.4 Physiological disorders Physiological disorders refer to the breakdown of tissues that is not caused by either invasion by pathogens (disease-causing organisms) or by mechanical damage. They may lead to premature deterioration and wastage of the tissue. Physiological disorders develop in response to an adverse environment, especially temperature or to a nutritional deficiency during growth and development. Many of these disorders occur as a result of prolonged periods of cool storage and/or levels of ethylene, carbon dioxide and oxygen. It is thought that they may be due to an imbalance in metabolism during cool storage. 28 Physiological disorders are characterised by the manifestation of abnormal appearance in specific parts of the fruit, which can be the peel, flesh or core area of the fruit. Susceptibility to induced disorders varies widely not only between varieties, but also due to orchard conditions, seasons and locality where the fruit is grown. Physiological disorders include superficial scald, core flush, brown heart, core and flesh browning. Low O2 and/or high CO2 concentrations can reduce the incidence and severity of certain physiological disorders such as scald of apples and pear, and chilling injury of some commodities (eg avocado, citrus fruits, chilli pepper and okra). 2.4.4.1 Superficial scald Superficial scald is a low temperature disorder involving browning of the skin. Although the tissue beneath the skin is not usually affected, the surface of scalded areas may be slightly depressed and wrinkled. Brooks et al. (1919) in their early work reported that superficial scald was due to the accumulation of toxic volatile products of the apple in the tissues of the fruit and in the surrounding air. They found that paper wraps impregnated with fats and oils reduced superficial scald in apples, with the chemical nature of the oils used being of little significance. Kidds and West (1933) however found that apples sprayed with oil scalded in the same way as untreated fruit. Huelin and Murray (1966) reported the presence of the sesquiterpene hydrocarbon, a-famesene, in the waxy coating of Granny Smith apples. It was later found that famesene and its oxidative products - conjugated trienes - accumulated in the coating of fruit during cool storage inducing scald. Huelin and Coggiola (1970a) postulated the sequence of scald development on apple fruit as follows: (i) the secretion of a-famesene by the epidermis and hydrodermis and 29 (ii) subsequent oxidation of a-famesene to conjugated trienes, which may then cause cell injury either by directly entering the cells or by polymerising to impermeable films (thus preventing gas exchange). Histological studies have shown that scald symptom begins with oxidative browning products and breakdown of cytoplasm in hypodermal cells (Bain, 1956). Bain and Mercer (1964) using the electron microscopy showed dense particles formed in the vacuoles of cells prior to any visible browning. These dense particles may precipitate membrane protein leading to tonoplast disruption, vacuolar and cytoplasmic mixing and "tanning". They suggested that these submicroscopic changes are consistent with the breakdown of the control mechanisms of the entire polyphenol system in both cytoplasm and vacuole. This suggests it is more likely that conjugated trienes enter the cells and attack the tonoplast to cause the subsequent "tanning" events rather than forming impermeable films. In addition, Anet (1974b) reported that transient free radicals evolved during the autoxidation of famesene are responsible for the cell damage. The incidence of superficial scald in fruit is closely correlated with the concentration of conjugated trienes (Huelin and Coggiola, 1970a,b,c). Apples as well as having natural wax also have natural antioxidants (Hopkirk, 1979). The concentration of the antioxidants was found to be highest at harvest declining during storage (Anet, 1974a). Anet therefore suggested that these natural antioxidants would protect the a-famesene from autoxidation early in storage. The addition of added antioxidants could be supplementing the natural antioxidants which Anet (1974a) found to increase in concentration in the first 2 - 3 weeks storage at 1°C. Hopkirk (1979) suggested that these natural antioxidants would minimise the level of development of superficial scald in fruit only if they could maintain the level of unsaturation in the external lipids of apples. 30 Chen et al. (1990) similarly observed a highly correlated relationship between scald incidence and conjugated trienes in 'd'Anjou' fruit and supported the theory that the accumulation of conjugated trienes induces scald. They reported a maximum increase in a-famesene content in the skin tissue of pears after 3 months of storage in air at -1°C, after which it declined. This decline was related to the oxidation of famesene to conjugated trienes, a process which was inhibited by diphylamine and ethoxyquin. The control of scald by these compunds was due to thier antioxidant action (ie inhibition of conjugated trienes biosynthesis). 2.4.4.2 Core and flesh browning This is a physiological disorder in which fruit Have a brown, soft core and surrounding flesh is also brown. Core browning can occur without flesh browning. According to Beattie et al. (1989), core browning of apples and pears occurs when fruits have been kept too long in the storage and the disorder develops quickly after the fruit has been removed from cold storage. Kader (1989) reported that concentrations of carbon dioxide above 5% could induce core and flesh browning in Bartlett pears. Nashi, like some other pome fruit is affected by core browning. The physiological life of Nashi core is believed to be shorter than the rest of the flesh. At the end of the natural storage life, the core of the fruit turns brown and black when senesces while the flesh remains unaffected (White et al., 1990). 2.4.4.3 Core flush Core flush like other physiological disorders develops after a period of storage. It appears as a visible pinkish or brownish discolouration of the core tissue. It has been suggested that core flush results from low temperature injury (Smock, 1946), carbon dioxide injury (Scott and Wills, 1976), or senescence (Lougheed et al., 1978). 31 Control methods presently recommended include harvesting apples at optimal maturity (Little et al., 1985) or storing them in controlled atmosphere, preferably with low oxygen and low carbon dioxide (Smock, 1946; Lougheed et al., 1978 and Little et al., 1985). Removal of ethylene from the storage atmosphere can also be beneficial (Little et al., 1985). 2.5 ATMOSPHERE MODIFICATION According to Burton (1974), if the useful storage life of a commodity is limited by biochemical changes - as may be the case mainly in materials that has the potential for rapid development or change when stored - then reducing the rate of change will increase the storage life. Since respiration is an index of the rate of metabolic turnover, reducing its rate could be considered as a possible means of slowing metabolism. Application of the Law of Mass Action to the simple concept of respiration suggests that if carbon dioxide in the storage atmosphere is augmented and the oxygen reduced, respiration should be reduced and storage extended (Burton, 1974). Certain postharvest techniques such as controlled and modified atmosphere storage utilise this principle in their application. Fruit respires and ripens in normal air which contains 78% nitrogen, 21% oxygen, 0.03% carbon dioxide, water vapour and inert gases. Atmosphere modification therefore, implies the addition or removal of gases, resulting in an atmosphere composition different from that of normal air. This is achieved by controlled or modified atmosphere storage; with their effects varying with the storage temperature, produce type and physiological age at harvest (Zagory and Kader, 1989). 32 2.5.1 Controlled atmosphere (CA) storage Controlled atmosphere storage implies the removal and/or addition of different gases into the storage area, effectively creating an artificial atmosphere most favourable for significantly prolonging the shelf life of the commodity. Pioneering work of Kidd and West (1927a,b) led to the development of controlled atmosphere storage which, in conjunction with refrigeration, has been used extensively to extend the storage life of apples and has been recommended for other fruits and vegetables (Geeson and Browne, 1980; Geeson, 1984). Although many fruit and vegetables respond to CA, the improvement in storage life is insufficient to warrant the extra cost since the technique involves high capital and maintenance costs. Controlled Atmosphere storage also has some other problems associated with it. Closely related species, different cultivars of the same species and tissue maturation variations often result in unpredictable oxygen and carbon dioxide tolerance limits (Haard, 1985; Kader, 1986). Physiological disorders induced by CA include internal browning and surface pitting of apples (Kader, 1986). Economically, CA storage units are costly to construct and maintain, and are limited or unavailable in many countries. In addition, commodities must be quickly used following the opening of the chamber. 2.5.2 Modified atmosphere (MA) storage Modified atmosphere storage, like CA, results in the alteration of the gaseous composition except that in this instance there is no close control of the gases (Wills et al., 1989). It is relatively simple and cheap when compared to the CA system, and allows for the extension of produce throughout a handling chain. Modified Atmosphere storage is achieved using physical barriers such as plastic films, plastic shrink wraps 33 (Ben-Yehosua, 1983, 1985; Pauli and Chen, 1989) and waxing/coating (Eaks and Ludi, 1960; Ben-Yehosua, 1967). 2.5.2.1 Polymeric films The use of polymeric films to achieve modified atmosphere has been either as box liners, storage bags or individual fruit shrinkwraps. Polymeric films ensure maintenance of humidity surrounding the produce (Ben-Yehosua, 1985) resulting in a slower rate of moisture loss. Although this storage technique seems simple, the right film of the appropriate thickness must be obtained and the ratio of surface area to mass of enclosed produce must be correct (Lowings and Cutts, 1982). Polyethylene box liners, either sealed or unsealed have been used for storing pears and apples (Hardenburg, 1971). Quality of Hass' avocado was improved with sealed polyethylene bags with and without low temperature storage (Oudit and Scott, 1973; Scott and Chaplin, 1978; Chaplin and Hawson, 1981). Unsealed or perforated bags are also used to reduce abrasion damage. The inclusion of an ethylene scrubber, potassium permanganate, in a sealed polyethylene bag enhanced the beneficial effects of MA packing of banana (Scott et al, 1970). Zagory and Kader (1989) recommended low density polyethylene and polyvinyl chloride films for use in wrapping fresh fruit and vegetables. Individual shrink-wrapping was found to extend shelf life of fresh apples (Anzueto and Rizvi, 1985) and also showed promising results in minimising losses in matured green tomatoes (Hulbert and Bhowmik, 1987). High density polyethylene film was effective in prolonging the storage life of grapefruits at temperatures above those causing chilling injury (Ben-Yehosua et al., 1983) and was suggested as a substitute for low temperature storage. A similar recommendation was made to store peaches at 10°C, a temperature higher than the normal storage temperature of 1-2°C, wrapped in polyolefin film 34 (Bhowmik and Sebris, 1988). Film wrapping reduced chilling injury and decay of 'Bearss' lemons stored at 1°C for 21 days (McDonald, 1989). Although individual seal packaging protects produce from infection of microorganisms from other infected fruit (Ben-Yehosua et al., 1981), it is more costly and permits the accumulation of ethylene and can result in stimulation of yellow colour in less matured apples (Heaton et al, 1990). Loss of crispiness was also reported as a problem in shrink-wrapped Starkrimson cultivar apples (Heaton et al., 1990). Production of acetaldehyde, ethanol and methanol, indicating anaerobic respiration; was reported in low density polyethylene film-wrapped cucumbers stored at 7°C (Risse et al., 1987). f 2.5.2.2 Commercial coatings Commercial coatings of horticultural produce involves the use of artificial surface coatings on fresh fruit and vegetables. Compounds that have been used as protective coatings include acetylated monoglycerides, natural waxes, mineral and vegetable oils, polysaccharides and protein; all of which are edible films or coating. The word coating is used synonymously with waxing, although waxing in most cases is used when the coating involved is a natural or artificial wax. 35 2.6 BIOCHEMICAL AND PHYSIOLOGICAL EFFECTS OF MODIFIED ATMOSPHERE It is assumed that gaseous exchange across the integument of fruits and vegetables occurs via pores (stomata/lenticels) which perforate their surfaces and therefore limited by the effective stomatal apertures. The restriction of gaseous diffusion through the stomata by coating results in the modification of the internal atmosphere of coated fruit. (Banks, 1984). The immediate effect of modified atmosphere is upon the biochemical reactions in which the constituents of the atmosphere participate. To reduce the rate of quality deterioration of stored fruits and vegetables, physical and chemical treatments of tissues are required to impede catabolic and anabolic processes (eg respiration, ripening and senescence) so that normal tissue structure and the "just- picked" chemical composition can be maintained as much as possible. Reduced O2 and/or elevated CO2 which decreases the rate of respiration is the primary purpose of controlled and modified atmosphere storage. Other factors involved in postharvest deterioration of fresh produce include metabolic changes (biochemical changes associated with respiratory metabolism, ethylene biosynthesis and action, and compositional changes); growth and development (anatomical and morphological changes); physical injuries; water loss; physiological disorders and pathological breakdown. 2.6.1 Effect on respiration Although O2 is not directly involved in the reactions of the Kreb cycle (as discussed in Section 2.2.1), the cycle can only operate under aerobic conditions in a commodity. This is because NAD+ and FAD in the mitochondrion can be regenerated only by the transfer 36 of electrons from NADH and FADH2 to O2 (in the oxidative phosphorylation pathway) with the formation of CO2. Studies of the effect of high CO2 levels on Krebs cycle intermediates and enzymes showed accumulation of succinic acid due to inhibition of succinic dehydrogenase activity in apples (Hulme, 1956; Knee, 1973; Monning, 1983), pears (Frenkel and Patterson, 1973) and lettuce (Brecht, 1980). Shipway and Bramlage (1973) found that CO2 levels above 6% stimulated malate oxidase and suppressed oxidation of citrate, a- ketoglutarate, succinate, fumarate and pyruvate in mitochondria isolated from apples. Ultrastructural changes (fragmentation, reduction in size and changes in shape of mitochondria), similar to those associated with senescence, was also observed in "Bartlett" pears stored at elevated CO2 (Frenkel and Patterson, 1977). It has been well documented that the rate of respiration of fresh fruits and vegetables is markedly affected by low O2 or high CO2 concentration in the atmosphere. The work of Kidd and West (1922; 1930) established that the onset of the climacteric rise in apples and pear was retarded by lowering the O2 concentration or raising the CO2 level. They found that the onset of the climacteric in apples was accelerated by oxygen tensions higher than in air. Singh et al (1954) reported a critical oxygen level of 9.2% for mangoes, below and above which CO2 evolution increased. The studies of Claypool and Allen (1946) with apricots, plums, peaches and pears indicated a decreased rate of CO2 output at oxygen tensions below air. Biale (1946) found that avocado, a fruit which exhibits a climacteric pattern of respiration, showed a reduced respiratory activity during the preclimacteric period when held under low oxygen. Within the range of 2.5 to 21% oxygen the time required to reach the climacteric peak was extended in proportion to the decrease in oxygen tension 37 and the intensity of respiration at the peak was reduced. Reduction of oxygen concentration from 16.6% to 7.4% in banana coated with Pro-long at 20°C resulted in a 14% reduction in the respiration rate (Banks, 1984). Solomos (1982) suggested that the decrease in respiration rate in response to low oxygen may be the result of a diminution of activity of an oxidase which have a 5-6 times lower oxygen affinity than cytochrome oxidase. Elevated CO2 concentrations also reduces the respiration rate of fresh fruits and vegetables and has a beneficial maximum limit before which accumulation of ethanol and acetaldehyde can occur within the tissues. Carbon dioxide concentrations of about 10% have been reported to result in formation of’aldehyde and ethanol in black currants (Smith, 1957), mango fruits (Lakshminarayara and Subramanyan, 1970) and citrus fruits (Davis et al., 1973) 2.6.2 Effect on ethylene production and action The rate of ethylene synthesis is directly related to O2 concentration. Reduced oxygen levels below 8% decreases ethylene production by fresh fruit and vegetables and also reduces their sensitivity to ethylene. Burg and Burg (1967, 1969) reported a halving in the production of ethylene and a retardation in fruit ripening when held under 2.5% O2. Elevated CO2 level can however reduce, promote or have no effect on ethylene production rates by fruit, depending on the commodity and the CO2 concentration. Increase in ethylene production by some commodities during and /or following exposure to CO2 was reported to have occurred only when the CO2 concentration was high enough to cause physiological injury to the tissue. It is not known however, whether this 38 high C02-stress-induced ethylene is due to partial shift from aerobic to anaerobic conditions or to other mechanisms. Chaves and Tomas (1954) observed a reduction in ethylene production and an increase in the ACC content of Granny Smith apples following exposure to elevated CO2 (20%) for 2 hours. This suggests perhaps elevated CO2 impeded the enzyme system (EFE) responsible for the conversion of ACC into ethylene. Burg and Burg (1967) interpreted from their kinetic studies that the binding affinity of ethylene to the receptor depends upon O2 and CO2; ie O2 increases the affinity, while CO2 reduces it. A halving of the binding of ethylene in air was reported when O2 was decreased to 3%. Oxygen is also required as a coupling activator for ethylene action (Burg and Burg, 1967), possibly relating to the oxidation of ethylene during action (Beyer, 1975). Although oxygen concentration may affect binding of ethylene to the receptor/oxidation site, kinetic data suggests that a high ethylene concentration should overcome the inhibitory effect of low oxygen (Burg and Burg, 1967). The inhibitory effect of low oxygen on ripening does not therefore arise from intermediary effects on ethylene production or action; low oxygen atmospheres must inhibit ripening changes, consequent upon action. The mode of action of CO2 in inhibiting or reducing ethylene effects in fruit and vegetable is not fully confirmed, but Burg and Burg (1967) suggested that CO2 acts as a competitive inhibitor with the ability to displace ethylene from the binding site of a metalenzyme. The competition between CO2 and ethylene is also explained in other physiological effects such as ethylene causing flowering fading, and CO2 reversing the 39 response and also delaying normal fading (Smith and Parker, 1966; Smith et ah, 1966); ethylene accelerating and CO2 delaying abscission; and fruit ripening is hastened by ethylene and delayed by CO2. Beyer (1981, 1985) however theorised that since ethylene at the receptor site can be oxidised by cellular enzymes to ethylene oxide, CO2 may act as a feedback or mass-action inhibitor. 2.6.3 Effect on compositional changes The effect of modified atmosphere is associated with a retardation of some of the processes involved in ripening. Rhodes (1970) defined the ripening of fruits as the sequence of changes in colour, flavour and texture which leads to a state at which the fruit is acceptable to eat. Some knowledge of tfie changes occurring during ripening and the effects of holding fruit in different gas mixtures is important in understanding the mechanisms by which coating exerts its effects. There are two principal theories concerning the mechanism of ripening in fruits. The first is based on the observed quantitative and qualitative changes in protein content and enzymic activity in ripening fruits (Rhodes, 1980). Frenkel et al (1968) found that protein synthesis was necessary for the onset of the increased ethylene production in pome fruits. The second theory invokes the breakdown of "organisation resistance" permitting the release or activation of bound forms of enzymes. Changes in intercellular compartmentation allows pre-existing enzymes access to substrates, cofactors, inhibitors and activators (Rhodes, 1980). One of the main benefits of MA storage is the prevention of ripening and the associated changes in fruit. Oxygen concentrations lower than 8% and elevated CO2 levels (above 1%) have significant effect on retarding fruit ripening (Kader et al, 1989). The effect of 40 the elevated CO2 was said to be additive to that of the reduced O2 atmosphere. The effect of MA on delay or inhibition of ripening is greater at higher temperatures, thus allowing the handling of ripening (climacteric-type) fruits at temperatures higher than their optimum temperature. A beneficial use for chilling-sensitive fruits such as tomatoes, melons, avocados, bananas, and mangoes, is expected, as low temperatures can be dispensed with completely. Many of the compositional changes which occur in harvested fruits and vegetables influence their colour, texture, flavour and nutritive value. Although some of these changes are desirable, others are detrimental to the quality of the commodity. t 2.6.3.1 Colour 2.6.3.1.1 Chlorophyll degradation The colour of produce can be attributed to the compartmentalised chlorophylls and carotenoids in plastids, and to anthocyanin in vacuoles of parenchyma cells. Oxygen may be directly involved in the alteration of the plastid structure, leading to chlorophyll breakdown. Chloroplast lipids are rich in linoleic and linolenic acids which are subject to peroxidation, either non-enzymic or catalysed by lipoxygenase. Knee et al. (1979) reported that the illumination of apples by visible light led to accelerated chlorophyll degradation, owing possibly to the photochemical enhancement of lipid peroxidation. Among the expected products of fatty acid degradation are hexanal and hexanal which maybe precursors of hexyl acetate, a dominant aroma compound of apples. Low oxygen storage was reported to have suppressed the synthesis of hexyl acetate (Patterson et al., 1974). 41 Loss of green colour (chlorophyll) and biosynthesis of yellow and orange colours (carotenoids) and red and blue colours (anthocyanin) were reported to have slowed down in fruits and vegetables kept in CA (Wankier et al. 1970; Wang et al., 1971; Isenberg, 1979; Smock, 1979). Magness and Diehl (1924) observed that skin coatings retarded the colouring of apples, and associated it with the increase in resistance of the skin to gaseous diffusion. Trout et al. (1953), reported very little change in colour over a period of 12 weeks in apples treated with coatings that were able to reduce the internal O2 level to less than 10 per cent. Biale and Young (1947) reported an extended storage life and delayed degradation of chlorophyll in matured green lemons by lowered oxygen levels whilst degreening of lime t was delayed under temperate and tropical ambient conditions by Prolong, a sucrose ester coating (Motlagh and Quantick, 1988). 2.6.3.1.2 Discolouration Phenolases, enzymes responsible for the oxidation of phenolic substrates and causing discolouration at cut surfaces, have a comparatively low affinity for O2 and are therefore susceptible to moderate changes in oxygen tension. Browning of the cut ends of broccoli (Smith, 1938) and lettuce (Singh, et al., 1972) was reduced or almost prevented by O2 concentrations of 5% or below as was the blackening of pre-peeled potato tubers. Yahia et al (1983) however, observed an increased brown discolouration of grapes with elevated CO2 concentrations (5 - 15%). 2.6.3 2 Texture Texture shows the physical state of a tissue. Structural and physical characteristics of the cell walls, cell turgidity, vacuolar size and fluidity, proportion of fundamental and 42 vascular tissues, and the tenacity of the middle lamellae between cells all contribute to the texture of fruit and vegetables. Softening of fruit tissue during ripening is important from the stand point of textural acceptability. The alteration of polymeric carbohydrates in the middle lamellae and the primary cell walls of ripening fruits is responsible for tissue softening. Softening is of great commercial importance since the postharvest life of the fruit is to a large extent limited by excessive softness, which brings with it an increase in physical damage during handling and an increase in disease susceptibility (Brady, 1987). • • • 9 The main components in the middle lamellae are pectic polysaccharides and the primary cell walls contain about 34% pectic polysaccharides. During ripening, the pectic polysaccharides in the middle lamellae of some fruits are thought to undergo polygalacturonase hydrolysis to form water-soluble pectic substances (Bartley and Knee, 1982) . Ultrastructural studies of apple and pear tissue indicated the dissolution of the middle lamellae during ripening (Ben-Arie et al., 1979). Correlations between the appearance of polygalacturonase enzymes and dissolution of the middle lamella was reported in avocado (Awad and Young, 1979), ripening tomatoes (Crooks and Grierson 1983) and stored apples (Han et al., 1985). Johnson (1982) suggested that raised levels of CO2 can improve fruit firmness when associated with O2 levels above 1%. Softening was retarded in apples by increasing concentrations (up to 12%) of CO2 and in some instances by reduced O2 (down to 2%) (Tomkins, 1965). 43 Firmness was generally best maintained by coatings with an appreciable resistance to CO2 (Trout et al., 1953), with coated apples been crispier and juicier than untreated fruit. Smith and Stow (1984) did not find the same marked effect on firmness with an external atmosphere containing 8% CO2, a level similar to that found in fruit treated with 3% sucrose ester coating. They therefore concluded that the effects of the coating might not be simply the consequence of the raised CO2 levels. 2.6.3.3 Effect on flavour Changes in carbohydrates, organic acids, protein, amino acids, lipids and phenols can influence the flavour of fruit and vegetables. Oxygen concentration has a marked effect on carbohydrate metabolism. Hansen and Rumpf (1973) noted that the loss of sucrose from stored carrots was less in low oxygen environments though the content of reducing sugars was lower than in air. High CO2 leads to an increase in citric acid in pears and a decrease in malic acid (Williams and Patterson, 1964). The development of a malate decarboxylating system in pome fruit was almost prevented by reducing the O2 concentration to 3% and entirely prevented at lower concentrations (Hulme and Rhodes, 1971). 2.6.4 Effect on physiological disorders Brooks et al. (1919) in their early work reported that superficial scald was due to the accumulation of toxic volatile products of the apple in the tissues of the fruit and in the surrounding air. They found that paper wraps impregnated with fats and oils reduced superficial scald in apples with the chemical nature of the oils used being of little significance. Kidds and West (1933), however found that apples sprayed with oil scalded in the same way as untreated fruit. 44 Soft scald is a physiological disorder of apples that leads to browning of the skin and flesh during low temperature storage of susceptible varieties. Lipids applied to fruit surface were observed to decrease the incidence of soft scald in Jonathan apples during cool storage (Wills et al., 1980). The mechanism by which lipids reduce the incidence of soft scald is not known but their principal action is suggested to involve indirect inhibition of hexanol synthesis, this compound being a toxic product of metabolism (Hopkirk and Wills, 1981). Application of antioxidants protects the fruit from some oxidation reactions, unrelated to fatty acid metabolism, which results in the accumulation of toxin and subsequent manifestation of soft scald symptoms. Lipids applied to apples may therefore be coating the naturally occurring antioxidants in the cuticle reducing the rate of their degradation. An alternative to the above mode of action for reducing soft scald is that coating the fruit may reduce the permeability of the skin to gas exchange therefore reducing the rate of metabolism of the fruit. This is also a common effect of modified atmosphere storage (Filder, 1973). Trout et al. (1940) found that storage in a modified atmosphere of 5% CO2 and 16% O2 reduced the level of soft scald in Jonathan apples. Hopkirk (1979), however did not observe a reduction in the respiration rate of apples treated with methyl stearate and methyl oleate in concentrations sufficient to reduce soft scald. Brown heart in apples is a brown discolouration within the vascular region developing in the senescent apple. This disorder probably follows the decreased permeability of the integument, and increased concentration of CO2 in the flesh, during normal storage in air. Application of an appropriate coating could reduce the metabolic activity of the fruit, thereby delaying senescence. 45 Although low O2 and/or high CO2 concentrations can reduce the incidence and severity of certain physiological disorders, levels outside the tolerance of some commodities can induce physiological disorders, such as brown stain of lettuce, internal browning and surface pitting of pome fruits and blackheart of potato (Kader et al., 1989). Brown heart, an internal disorder caused by excess CO2 was reported in Sturmer Pippin apples treated with 15% C.O.S (castor oil-shellac aicoholic solution) and stored at 10°C (Hall et al. 1953 ). Core breakdown was also re,ported in pears. Both disorders are associated with succinic acid accumulation (Hulme., 1956; Williams & Patterson, 1964) probably related to the effect of increased CO2 r 1957). In other words, elevated CO2 reduces the activity of the oxidase subsequently resulting in the accumulation of succinic acid. Pink or brown discolouration has been observed in mushrooms stored at low temperatures in enhanced CO2 concentrations (Smith 1964, 1965). 2.6.5 Effect on growth and development Fruits and vegetables continue to grow and develop after harvest. This may, however, cause undesirable effects. Sprouting of potatoes, onions, garlic and root crops accelerates deterioration reducing their utilisation value. Asparagus spears also continue to elongate and curve, becoming tougher and less palatable. Burton (1974) stimulated the sprouting of potatoes at 10°C by increasing the concentration of ambient CO2 to 2 - 4%. Sprouting of potatoes was inhibited by exposure to 15% CO2 at 10°C but encouraged in reduced O2 (2-4%) atmospheres. Sprouting and rooting were inhibited when onions w/ere kept in 3% O2 + 5 - 10% CO2 at 1°C (Isenberg, 1979). Lowered O2 was also reported to have inhibited sprouting and rooting in carrots, but increased decay problems. 46 Elongation of cut asparagus and opening of the mushroom caps were also inhibited by decreased O2 and/or increased CO2 (Murr and Morris, 1974; Burton, 1974; Isenberg, 1979). Although, the mechanism involved is yet to be explained, Isenberg (1979) suggested that controlled atmosphere may influence the levels of endogenous growth regulators. 2.6.6 Effect on decay microorganisms Modified atmospheres can have direct and indirect effects on postharvest pathogens. El- Goorani and Sommer (1981) pointed out that delaying senescence, including fruit ripening, reduces susceptibility of fruits and vegetables to pathogens. Modified atmosphere conditions unfavourable to a given commodity, on the other hand, can induce physiological breakdown and render the commodity more susceptible to pathogens. El-Goorani and Sommer (1981) reported that O2 levels below 1% and/or CO2 levels above 10% are needed to significantly suppress fungal growth while elevated CO2 levels (10 to 15%) can be used to provide fungistatic effects on commodities that tolerate such high CO2 levels. 2.7 EDIBLE COATINGS 2.7.1 Principle of edible coatings Since the principles of gas storage were first advocated in the late 1920's (Kidd & West, 1927), several attempts have been made to find coatings which would mimic the effects of CA storage by allowing differential permeation of O2, CO2 & H2O vapour through the coating. Thus, in order to mimic the effect of CA storage, the coating would have to act as a selectively permeable barrier in such a way that oxygen levels in the fruit could be markedly lowered without a concomitant excessive rise in CO2 levels during storage. 47 Suitable coating would result in a type of modified atmosphere storage, operating at the level of the individual fruit, by reducing the permeability of the skin to gases. Films and coatings applied to fruit surfaces modifies the availability of oxygen and traps the carbon dioxide in the fruit, slowing down their respiration by retarding ripening and altering the level of other physiologically active compounds (Hall et al., 1953), hence extending their edible life. Kidd and his coworker (1927a,b) observed a reduction in the rate of respiration, which correlated with an extension in storage life during modified atmosphere storage of apples. The degree of these effects on a given commodity, will however, depend on species, cultivar, surface-to-volume ratio and respiration rate (Platenius, 1939). t 2.7.2 Historical background Some hydrophobic substances such as beeswax, paraffin wax and some polymers have been used as coating material to enhance the keeping qualities and appearance of fresh produce. The practice of applying waxes to fruit after harvest is quite ancient. According to Kester and Fennema (1986), Hardenburg (1967) dated the use of molten waxes as coatings for orange and lemons back to the 12th or 13th century in China. Plant physiologists have long known that a thin film of natural or artificial wax on the surface of plant tissue is effective in reducing its rate of water loss and wilting (Platenius, 1939). Platenius dated the commercial use of wax coatings on many types of perishable produce in the United States to 1919; with the citrus industry been the first to recognise the advantages of the waxing method. The possibility of using wax emulsion on vegetables was first suggested by Harvey and Landon (1935), who reported that waxing reduced the water loss of winter squash by 50%. Waxing of banana and avocado was successful but the benefit did not cover the cost and was, therefore, abandoned in Australia (Scott, personal communication). 48 Although wax emulsions have been extensively used in the citrus industry, their limitations include being expensive and unaccessible to farmers in most developing countries. Wax dissolves in organic solvents such as alcohol, making the production of wax emulsion more expensive, especially in countries with a high duty on alcohol. Fats and oils, because of their hydrophobic nature, may provide an alternative means of prolonging storage life that is cheaper and requires less capital outlay. Utilisation of fat to enrobe food products has been long recognised in different food industries. These include confectionary products (eg chocolate coatings), meats, breakfast cereals as well as fresh produce (eg coating of fruit). According to Hulme (1949) preliminary work conducted in 1939 by the British Food Investigation Organisation confirmed that oil emulsion dips on English Cox's Orange Pippin apple reduced loss of weight, rate of respiration and ripening as evidenced by the change in colour and hardness. With the 1980s, came the extensive use of a variety of polysaccharides, proteins and lipids, utilised either alone or in mixtures, to produce composite films. Some of these include "Sonsyl" (manufactured by Gist-Brocades, Holland), Tal-Prolong (Lowings and Cutts, 1982), Semperfresh and Nutri-Save (Elson et al., 1985). "Sonsyl" reduced the rate of ripening of banana fruit ripened with ethylene (Banks 1984) but had several commercial disadvantages. It could only be supplied in a liquid formulation, which increased the cost of transportation and there were frequent problems with microbial contamination and decomposition of the coating concentrate (Banks, 1982). The coating lipid was also observed to foam excessively during mechanical application, with coated fruit having an undesirable tacky, glossy finish. 49 "Pro-long" is a powdered formulation of sucrose esters of fatty acids with an added salt of carboxyl methycellulose polysaccharide (Lowings and Cutts, 1982). Its mode of action involves the creation of a selectively permeable barrier that creates internal atmospheres which preserve the fruit and also reduces water loss and chilling injury. These are characteristics which might be utilised both in the storage of fruit and for the maintenance of quality throughout the marketing period (Banks, 1982). 2.8 MODIFIED ATMOSPHERE EFFECT OF EDIBLE COATINGS ON FRUIT AND VEGETABLES 2.8.1 Water loss Water loss results in direct quantitative losses (loss of saleable weight), as well as losses in appearance (due to wilting and shrivelling), texture (softening, flaccidity, limpness, loss of crispness and juiciness) and nutritional quality (Wills et al., 1989). According to Kaplan (1986) fruit loses moisture rapidly after washing (ca. 6-8% of harvest weight) when held under normal holding conditions. Washed fruit also softens, with a dull appearance and increased susceptibility to infection. Ben-Yehosua (1967) reported that oranges disinfected in S.O.P.P and rinsed in water lost weight and volume at a significant higher rate and received lower quality scores than untreated fruit. The mean storage life (at 20°C and 65 - 85% RH) was 4-5 weeks for untreated fruit and 3 weeks for treated ones. Tompkins (1965) observed that the use of ethyl alcohol as a fungicide resulted in shrivelling and more rapid deterioration in fruit. To overcome this, a mixture of castor oil and shellac was added to the alcohol to replace the natural wax, most of which had been removed by the alcohol. 50 Waxes which have been used to retard desiccation include paraffin wax, beeswax, camauba wax, candelilla wax, and rice bran wax (Warth, 1956; Paredes-Lopez et al., 1974; Lawrence and Iyengar, 1983; Kaplan, 1986). Experiments with citrus fruits indicates that application of a selected artificial wax does not produce off-flavours while reducing moisture loss (Ben-Yehosua, 1967). Paredes-Lopez et al. (1974) reported reduction in weight loss of about 40% in limes coated with 15% candelilla wax. This was in addition to the attractive gloss imparted to the fruit. Although excessive water loss from some produce does not necessarily affect' their edibility, a certain degree of wilting renders them less attractive to the consumer. Shrivelling may not be a problem if it is feasible to have relative humidities as high as 95 to 98 per cent in the cold rooms. The advantage of waxing is that it reduces shrinkage losses and lowers the rate of transpiration and wilting, not only during storage, but throughout the subsequent marketing period. Enrobing an entire product with an edible film of coating restricts movement of moisture between the product and its surrounding atmosphere. Edible waxes are significandy more resistant to moisture loss than most other lipid and non lipid films. Schultz et al (1949) evaluated the water vapour-barrier properties of lipids and observed that waxes were the most effective in blocking moisture migration, with paraffin wax being the most resistant followed by beeswax. Similar reports were also reported by Landmann et al (1960) and Kamper and Fennema (1984a). The great resistance of paraffin and beeswax coatings to diffusion of water vapour has been related to their molecular compositions. Paraffin wax consists of a mixture of long-chain, saturated hydrocarbons (Warth, 1956), while beeswax comprises 71% hydrocarbons and 8% long-chain fatty acids, with the remaining 6% unidentified. The absence of polar 51 groups in paraffin and the relatively low level in beeswax accounts for their hydrophobic characteristics (Swem, 1964). 2.8.2 Appearance The use of wax as a coating imparts a brilliant shine on the produce. Unlike citrus which are waxed after harvest to extend their shelf life, apples are waxed after storage simply to make the fruit more shiny. Kaplan (1986) reported that washed citrus preserved by a wax film had a fresh, bright, attractive appearance; as well as a high internal quality and freshness. Platenius (1939) observed that waxed carrots had an improved colour even though the wax itself did not contain any pigments. Emulsions, particularly those containing a high proportion of camauba wax, were also reported to give an attractive gloss to cucumber and tomatoes. 2.8.3 Ripening and quality maintenance Polyethylene wax emulsions have become important in the storage and marketing of avocado because they reduce moisture loss, delay softening and improve appearance, especially of the 'Fuerte' cultivar (Lunt, et al., 1981; Durand et al., 1984). Tag (developed by the Plastics Department of Weizmann Institute of Science and produced by Makhteshium Chemical Company, Beer Sheva, Israel) extended the storage life of banana by delaying and inhibiting flecking and reducing decay of the cut surfaces of the fruit (Ben-Yehosua, 1966). It also extended the storage life of orange fruit by 100% and was effective over a wide range of conditions (23-31°C, 40-50% RH) (Ben-Yehosua, 1967). The storage life was related to the effect of the coating in preventing the peel from drying rather than to its effect on the internal atmosphere. 52 Durst (1967) successfully used natural materials such as vegetable oil emulsions and melted lard emulsions to coat fig bar. Ukai et al (1976) used hydrophobic emulsions to coat fruit and vegetables such as orange, peas, apples, green beans, tomatoes, pears and peaches. Beeswax-coconut oil emulsion extended the shelf life of peaches by 4 days (Erbil and Muftugil, 1986). The emulsion decreased water vapour and O2 gas transfer and resulted in a diminished respiration rate . Prolong/Semperffesh has been used to extend the storage life of bananas (Banks, 1984), plantain (Olorunda and Aworh, 1984) apples (Smith and Stow, 1984; Drake et al 1987; Chai, et al., 1991), limes (Motlagh and Quantik, 1988) mango (Dhalla and Hanson, 1988) and some varieties of pears (Somsrivichai et al., 1990). An edible composite coating, "USDA", was developed at the US Department of Agriculture. It retarded ripening in some climacteric fruit such as tomatoes, mangoes, papaya and bananas (Nisperos-Carriedo et al., 1991). The anti-browning property of the coating was further improved by the incorporation of an antioxidant and a chelator. 2.8.4 Physiological disorders Lipids generally have a relatively low polarity and are therefore used to block transport of moisture. They have, however, been reported to serve other functions such as reduction of surface abrasion during handling (Hardenburg, 1967) and control of "soft scald" formation in stored apples (Wills et al 1980). Lipids applied to the fruit surface either in an ethanol solution or an oil-in-water emulsion decreased incidence of soft scald in Jonathan apples during cool storage (Wills et al., 1980). These compounds (lipid) included methyl esters of lauric, palmitic, stearic, oleic, linoleic and linolenic acids, as well as palm oil, sunflower oil, safflower oil, coconut oil, lard and lecithin. 53 Mineral oil and waxes applied to the surface of apples were also reported to have controlled superficial scald (Shutak and Christopher, 1953, 1956; Shutak et al., 1953; Smock, 1957; Porritt and Meheriuk, 1970), bitter pit (Hall et al., 1953, Adams, 1969), core flush (Meheriuk and Porritt, 1972) and Jonathan spot (Hall et al., 1953). The use of Prolong as a surface coating reduced core breakdown in "Bartlett" pear and superficial scald in'd'Anjou' pears (Meheriuk and Lau, 1988). Chilling injury, a low temperature disorder resulting from the exposure of susceptible plant tissues ( especially those of tropical and subtropical region) to temperatures below 15°C, has also been reduced by coatings. Jones et al (1978) found that safflower oil and t mineral oil effectively reduced chilling injury in bananas stored at 9°C. Immersing grapefruit in vegetable oils and vegetable oil-water emulsions prior to storage at 3°C markedly delayed and reduced symptoms of chilling injury (Aljuburi and Huff, 1984). Certain rind disorders, such as 'pitting', sunken areas usually associated with cold injury and 'aging', rind tissue collapsed near the stem end, have been reduced substantially by polyethylene emulsion coatings (Davis and Harding, 1960) and solvent-type wax (Chace et al., 1969). 2.8.5 Decay organism Coating Valencia orange with solvent wax controlled decay by 83.1% after 2 weeks storage at 21°C (Newall and Grierson, 1955). Flecking, caused by Anthracnose, on banana was inhibited on banana coated with Tag (Ben-Yehosua, 1966). Pungent mustard oil has been used to control rot causing fungal pathogens such as Trichothecium roseum and Rhizopus stolonifer in apples (Kaul and Munjal, 1982). Coating serves as a barrier between the microorganims (preharvest infection) and the atmosphere, depriving the organism of needed growth conditions (Kotze and Kucschke, 54 1978). Darvas et al (1990) on the other hand suggested that the microclimate condition created by coatings favours the developments of postharvest diseases. 2.8.6 Gaseous exchange and respiration rate The immediate effect of a coating has always been interference with the normal gas exchange through the epidermis, resulting in increased resistance to O2, markedly reducing the O2 available to the tissue for respiration (Trout et al 1953). The reduced respiration correlated with the decreased O2 concentration, but not with the internal CO2. Trout et al (1953) observed that resistance to O2 and CO2 varies with type of oil or wax. Castor oil was reported to have greater resistance to O2 than shellac, which had greater resistance to CO2. Emulsions (10%)' of four different oils (castor, paraffin, linseed and cottonseed) and shellac reduced respiration of Granny Smith apples by about 45% while the reduction by castor oil and shellac in alcohol (C.O.S) was only 32% (p<0.01). Platenius (1939) found that air and CO2 diffuse through moist wax films at rates directly proportional to their solubilities in water. The diffusion rate was very low when both the membrane and the gases were dry. This indicated that the diffusion of gases depends primarily on its solubility in the continuous soap (wet) phase of the film, rather than on the porosity of the entire membrane. Trout et al (1953) verified this by comparing the effect of alcohol solutions and aqueous emulsions of castor oil, shellac and castor oil plus shellac on the respiration rate of Granny Smith apples. The alcohol solutions reduced the respiration rate by 25 to 30 % and the aqueous emulsion by only 18 %. As the resistance of the emulsion to gases was less than that of alcohol solutions, the continuous aqueous soap phase is an important factor in regulating diffusion of gases through emulsion coatings. 55 It is possible to have a high respiration rate even with a very low O2 concentration especially if respiration is expressed in terms of CO2 output. The low O2 concentration shifts the respiration from aerobic to anaerobic increasing the respiratory quotient. Platenius (1939) also found that there was a decided lowering of O2 consumption in waxed carrots while the rate of CO2 production remained practically unchanged. He proposed that CO2 accumulated within the tissue soon after waxing, with such an increase in concentration resulting in a rise in the concentration gradient between the two sides of the wax film. This in turn would have caused a corresponding increase in the outward diffusion of CO2. Smock (1936) observed injury to apples and pears coated r with wax emulsions. The difference in the response of pears and apples, and carrots to wax reported by the two workers can be explained by the respiratory quotient normally existing in different plant tissues after harvest. Magness and Diehl (1924) found that the ratio of CO2 production to O2 uptake in apples and pears remain close to 1. Platenius (1939), however, reported the respiratory quotient of carrot was above unity initially, but decreased rapidly to 0.55 after 5 days. Tolerance levels of O2 and CO2 for each commodity (Table 4 & 5) has to be maintained to prevent a shift from aerobic to anaerobic respiration and the consequent accumulation of ethanol and acetaldehyde causing off-flavours. 56 Table 4. Classification of fruit and vegetables according to their tolerance to low oxygen concentrations. Minimum O2 concentration Commodities tolerated (%) 0.5 Tree nuts, dried fruits and vegetables 1.0 Some cultivars of apples and pears, most cut or sliced fruits and vegetables. 2.0 Most cultivars of apples and pears, kiwi fruit, apricot, nectarine, peach, plum. 3.0 Avocado, tomatoes, pepper, cucumber. from Kader et al., (1989) t Table 5. Classification of fruits and vegetables according to their tolerance to elevated carbon dioxide concentrations. Maximum CO2 concentration Commodities tolerated (%) 2 Apple (Golden delicious), Asian pear, European pear apricot, tomato, pepper (sweet) 5 Apple (most cultivars), peach, nectarine, plum, mango orange, avocado, banana, carrot. 10 Cucumber, from Kader et al., (1989). 57 2.8.7 Detrimental effect of edible coatings One problem with coating fruits and vegetables is that the interference with the normal gas exchange in the produce may be too extreme and could result in anaerobic respiration and the development of undesirable flavours (Dhalla and Hanson, 1988). Platenius (1939) observed symptoms of internal breakdown, caused by suboxidation, in rutabagas treated with a hot - paraffin dip. Cucumbers treated by the same process underwent complete deterioration within 2 days. Smock (1936) observed definite injury to apples and pears treated with wax emulsions at high concentrations. Functional diseases such as brown heart and in some cases lenticel spotting were confirmed on English Cox's Orange Pippin apple treated with more concentrated (21%) oil emulsions t (Hulme, 1949). Adverse ripening was observed in Bartlett pear (Meheriuk, 1988). "Dodo" (a Nigerian food) made from plantain dipped in Pro-long had a poorer flavour score than those made from untreated plantain. This according to Olorunda and Aworh (1984) was due to a lower sugar content relative to the untreated fruit at a similar stage of ripeness, as determined by the colour index and pulpipeel ratio. Suitable combinations of waxes in appropriate concentrations, may influence the gas exchange of the waxed produce in such a way as to actually reduce the normal rate of chemical changes within the produce without detriment to the produce. Vegetables treated with wax emulsions (Brytene 333-B : high in paraffin content and Brytene 284-D : chiefly camauba wax) of low to medium concentrations showed no visible evidence of injury nor presence of alcoholic or other undesirable flavours (Platenius, 1939). A successful result was obtained with lower concentrations (12 & 14%) of oil emulsions on apple (Hulme, 1949). This however does not rule out the possibility of waxing resulting in harmful effects under certain conditions. Platenius (1939) found that different concentrations of wax emulsions had different effects on the 58 different vegetables used. This may be due to the fact that the respiration rate after harvest varies gready in different vegetables, with storage temperatures and the period of storage. In summary, types and concentrations of emulsions that will give a desirable effect on different produce will therefore depend on the respiratory quotient of the produce. A coating that is desirable on a produce stored at a high temperature may require a different concentration if intended for storage at a low temperature. Specific emulsions or coatings need to be developed for particular produce to avoid serious respiration changes and/or ripening problems. 2.9 DEVELOPMENT OF LIPID BASED EDIBLE COATINGS Earlier studies with non-water soluble "edible" coatings often resulted in off flavours, injury and internal breakdown, particularly when fruits were exposed to high temperatures. Ben-Yehosua (1967) discovered that oranges thickly coated with Tag had off-flavours resembling those of fruit held in an anaerobic atmosphere; while very thin coatings were not effective enough to achieve the desired responses. Various oils and waxes have different effects on extending the storage life of produce. Castor oil has been reported to have a greater resistance to O2 while shellac a greater resistance to CO2 (Trout et al., 1953). Combination of two or more oils/waxes could achieve an effective coating. Off-flavour in fruit, which is generally associated with anaerobic respiration caused by accumulation of CO2, could be stopped by using coatings which are semi-permeable to the respiratory gases. Oils could therefore be 59 emulsified to create a water phase between oil droplets for easier, but restricted, gaseous exchange between the fruit and the environment. The effect of a given coating usually increases with concentration and decreases with increasing alkalinity (Trout et al., 1953). Balancing the effectiveness and stability of an emulsion is very important. Waxing has been reported to control shrinkage during storage and improve appearance of fruit but conversely calyx browning and rots were observed on apples treated with wax emulsions, with the severity depending on the alkalinity and concentration of the emulsion (Trout et al., 1953). Waxes, being more difficult to emulsify than oils, require higher concentrations of soaps to produce a stable emulsion which would completely wet a fruit. 'Addition of shellac to paraffin wax was reported to have enabled the emulsion to wet the fruit completely, without the disadvantages due to use of additional soap (Trout et al., 1953). Trout et al. (1953) carried out several experiments using various oils and waxes as coatings. They reported that weight loss in Granny Smith apples increased by coating with emulsions of peanut oil, maize oil and the tighter mineral oils. Application of waxes and castor oil emulsion also reduced weight loss, the effect increasing with the addition of shellac. Paraffin wax was the most effective of the waxes used. Camauba and beeswax were less effective. Aqueous emulsions of all mineral oils tested, except high viscosity medicinal-grade paraffin oil, severely injured the skin, increased shrivelling and were less effective in retarding ripening. Special wax-free shellac gave better results than ordinary shellac which contains some lac wax and also a tittle orpiment (arsenic trisulphide). The wax-free shellac was completely soluble in alcohol, forming a brighter, more uniform and more effective coating. Castor oil retarded ripening and reduced wastage but increased wilting and was too oily and sticky on the apple. 60 The following conclusions were drawn from this work : i) Oil emulsions should contain 4-5% oil. ii) Wax emulsions should contain 6% wax. iii) Addition of shellac or similar material to emulsions decreases the concentrations to be used, and also reduces the excessive oiliness of oil emulsions. iv) Resistance to the passage of water vapour is in the order of: paraffin wax > beeswax > camauba wax > medicinal paraffin oil > shellac > vegetable oils > lighter mineral oils. v) Type of emulsifying agent used is very important. vi) In contrast to the effect on weight loss, oils were more effective than waxes in varying the internal atmosphere and reducing respiration. vii) Ten percent (10%) emulsion of mixed waxes (paraffin + lac/camauba wax) with a mildly alkaline soap (eg ammonium or morpholine) retarded ripening, maintained palatability and gave a bright natural appearance. viii) Storage temperature changes could be a problem in coated fruits. Coatings suitable for low temperature storage were often unsuitable when fruit are moved to higher temperatures. 2.10 CHEMISTRY AND PROPERTIES OF LIPID COATINGS 2.10.1 Fats and oils These are naturally occurring esters of glycerol and fatty acids; called triglycerides or simply glycerides. Each hydroxyl group of the trihydric alcohol glycerol is esterified to a fatty acid. The hydrophilic nature of fats and oils gives them a considerable quality of reducing moisture loss in produce, as well as restricting gas movement. 61 2.10.1.1 Chemical properties Rl, R2 and R3 represent the alkyl chain of the fatty acid. CH2OH Rl - COOH H2C0C(0)R1 CHOH + R2 - COOH HC0C(0)R2 CH2OH R3 -COOH H2C0C(0)R3 glycerol fatty acid triglycerides The two basic parts of a triglyceride molecule are the glyceryl portion and the fatty acid radicals. The combined molecular weight of a triglyceride varies with different fats from about 650 - 970 with the former portion (glyceryl) being 41. In other words the fatty acid contributes 94 - 96% of the total mass of a triglyceride molecule. They are of great influence both to the physical (hydrophobic character) and chemical character of the glycerides, particularly from the stand point of stability. Fats and oils of commercial importance have the fatty acids as a straight chain and nearly all contain an even number of carbon atoms. The alkyl chain is unsubstituted except for castor oil which contains the hydroxyl substituted acid, ricinoleic acid. Most fats and oils are based on Cl6 and Cl8 chained-acids with 0 to 3 ethylenic bonds except for coconut oil, which is rich in shorter chain acids and some marine oils such as fish liver oil, which contain acids with as many as 22 or more carbon and 6 or more ethylenic linkages. 62 Saturated fatty acids have higher melting points and represent the main constituents of solid fats eg lard and butter. Unsaturation lowers the melting point of fatty acids and fats. Unsaturated fatty acids are present in large amounts in some of the oils derived from plants eg oleic acid in olive oil and linoleic and linolenic acids in linseed oil. 2.10.1.2 Physical properties 2.10.1.2.1 Melting point The melting point increases with the chain length of the fatty acid. Thus, a short-chain saturated fatty acid such as butyric acid will have a lower melting point than the saturated fatty acids with longer chains and even some of the higher molecular-weight i unsaturated fatty acids, such as oleic acid. This, therefore, explains why coconut oil, which is almost 90% saturated fatty acids but has a high proportion of short chained fatty acids, is liquid at 36°C, whereas lard, which contains about 37% saturated fatty acids with longer chains, is solid at about the same temperature. For a given fatty acid chain length, saturated fatty acids have higher melting points than those which are unsaturated. 2.10.1.2.2 Oiliness and viscosity One of the noticeable characteristics of oils and fats is their "oiliness" or ability to form lubricant films, and they are quite similar to long-chain hydrocarbons in this respect. Viscosity of oils decreases slightly with an increase in unsaturation, thus viscosity is increased slightly by hydrogenation. Oils containing fatty acids of low molecular weight are slightly less viscous than oils of equivalent degree of unsaturation containing only high molecular-weight acids. Rapeseed and other erucic acid oils are a little more viscous than equally unsaturated oils composed of Cl6 and Cl8 acids. Castor oil is much more viscous than other oils, such 63 as sunflower, because of its high content of ricinoleic acid which readily forms intermolecular hydrogen bonds. Fatty oils show less change in viscosity with temperature than do mineral oils. 2.10.1.2.3 Surface and interfacial tension Surface tension increases with increase in chain length and decreases with an increase in temperature (Table 6). Interfacial tension is lowered in commercial fats by the presence of monoglycerides, phospholipids, free fatty acids or traces of soaps. Table 6. Surface tension of some oils at different temperatures (Swem, 1964) Surface tension (dynes/cm) Oil Temperature (°C) Cottonseed Castor Coconut 20 35.4 39.0 33.4 80 31.3 35.2 28.4 130 27.5 33.0 24.0 2.10.1.2.4 Solubility in water Fats and long-chain fatty acids have a low solubility in water, which decreases with an increase in chain length and decrease in temperature. It is therefore assumed that the longer the chain the lower the solubility in water, so short chained fatty acids maybe more likely to allow passage of water than long chain fatty acids. 64 2.10.1.3 Classification, sources and properties of lipids The individual property of lipids is important in assessing their suitability as coating on fruit and vegetables. The physical and chemical properties are determined to a large extent by the types of fatty acids precent in the glyceride. A summary of the properties of some lipids relevant to coating technology is presented in Table 7. Table 7. Classification, sources and properties of some commercially important lipids Lipid Source Properties Animal origin Butterfat cow's milk Has a distinctive flavour and aroma considered objectionable or distasteful when it becomes rancid. The main fatty acids are oleic, stearic, palmitic, and myristic acids. Lard fatty tissues of hogs - Vegetable origin Coconut oil Coconut flesh Liquid to approximately 28°C even though it has 85 to 95% saturated fatty acids. A high level of saturated fatty acids makes it resistant to oxidative changes (rancidity) under normal storage conditions. Sunflower oil sunflower seed Has a good flavour stability without a need for hydrogenation, Safflower oil safflower seed High linoleic 75 - 80% acid content, which is the highest of any known oil. Expensive and lack flavour stability. Olive oil olive tree More stable against oxidation than most liquid oils because of its low content of linoleic acid. 65 Table 7 (contd.) Almost completely lacking in drying properties or any tendency to become gummy when exposed as films. Palm oil palm tree Stable against oxidation and has no drying properties. Com oil com germ High level (about 55%) of polyunsaturated fatty acids. Low cloud point and melting point. Good keeping quality, but more expensive. Peanut oil peanut Sticky to touch. Rapeseed oil seeds of Brassica More viscous than ordinary oil. (canola) campestris or Napus Soybean oil soybean Preponderance of unsaturated fatty acids. Used extensively in the manufacture of drying oil products. Castor oil Can be converted to a conjugated acid oil, similar to tung or oticica oil, by dehydration process. Dehydrated castor oil is extensively used in the protective coating industries. Castor oil has also been used on fruit to retard ripening and reduce wastage. Has a greater resistance to O2 than CO2, but too oily after application and later became sticky (Trout et al 1953). Mineral oil Liquid paraffin 66 2.10.2 Emulsion and emulsifiers 2.10.2.1 Definitions An emulsion is a mixture of two or more immiscible liquids, for example water and oil. In any emulsion, the suspended droplets are referred to as the dispersed or internal phase while the medium in which they are suspended is the external or continuous phase. Emulsifiers, also known as surface-active agents or surfactants, are the interfacial components used to improve miscibility between the two or more solutions. They help stabilise the emulsion formed by lowering the interfacial tension between water and the other liquids. This change in the interfacial tension will cause the formation of a physical barrier around each droplet so as to impede their coalescence. 2.10.2.2 Types of emulsion The volume relationship between the two liquids determines the type of emulsion, and usually the liquid present in the lesser quantity would tend to become the dispersed phase. This however is not always the case. The question of which liquid is dispersed is determined by surface tension considerations, which are definitely related to the nature of the emulsifying agent. According to the accepted theory relating to emulsion type, the film created by the oriented surface-active molecules between the two phases of the emulsion is regarded as a 3rd phase which has separate surface tensions against each of the two liquids forming the emulsion. If a film has a greater affinity for water than oil, the surface tends to curve in such a direction as to reduce the total surface tension. The curve is formed with the area of low surface tension on the outside and that of high surface tension on the inside; and in this case it will tend to enclose globules of oil in water. On the other hand, if the film has a 67 greater affinity for oil than for water, the tendency will be to form an emulsion of water in oil. There are basically two types of emulsions : 1) The oil dispersed in water (o/w) type, which generally conducts electricity, can be diluted with water, feels more like water, dries rapidly, can be washed off the skin and exhibits the aqueous properties of the continuous phase; and 2) The water dispersed in oil (w/o) type, which is a poor conductor of electricity, may be diluted with solvents or oil, generally feels more like oil than water, resists drying or loss of water, difficult to remove by washing, and depending upon the oil phase, resembles the properties of the continuous phase. O/W are generally white while w/o are darker. A drop of an o/w emulsion will disperse in water while that of w/o remains unchanged (usually floating on the surface). 2.10.2.3 Emulsifier The creation of a bonafide food emulsion involves disrupting the dispersed phase into droplets of colloidal size, and protecting the newly formed droplet interface from immediate coalescence. In other words, with emulsion formation in foods, there is the need for stability - both transient (at the time of formation) and long-term (for product shelf life). In a water-oil emulsion the dispersed particles are constricted to a spherical form by surface tension. Surfactants that are particularly good emulsifying agents lower the interfacial tension between the two liquid phases. Since the interface must be extended to produce an emulsion, lowering the interfacial tension is favourable both to the formation and stabilisation of emulsions. 68 2.10.2.4 Types of emulsifiers 2.10.2.4.1 Oil and fat derivatives Fatty acids are particularly suitable for the production of surface active agents because of their specific and reproducible molecular structure. Commercially available fatty acids consist for the most part of normal or unbranched hydrocarbon chains of 12 to 18 carbon atoms, terminating in an active carboxyl group. When the terminal hydrogen atom is substituted with an alkali metal or when the carboxyl group is otherwise converted to a group of hydrophilic nature, the resulting molecule becomes endowed with special properties. Hydrophilic groups can also be introduced at the double bond of oleic acid f or at the hydroxyl group of ricinoleic acid. The hydrocarbon end will have an affinity for oils, aliphatic hydrocarbons, and similar water-insoluble compounds, whereas the opposite end of the molecule will be attracted to water or aqueous solutions. The two ends of the molecule will be sufficiently separated for the two affinities (hydrophobic and hydrophilic) to come into simultaneous and independent action. The size or length of the molecule is critical because there must be a balance between hydrophilic and hydrophobic properties. Too long a chain will cause an imbalance in the direction of too great an affinity for oily materials and little or no affinity for water which is shown by limited solubility in water. If the chain is too short, the compound will not be very surface active because of insufficient hydrophobic qualities and lack of colloidal 69 characteristics. In general the optimum chain length lies in the same range as that for the common fatty acids, ie 12 to 18. 2.10.2.4.2 Hydrocolloids Hydrophilic colloids or hydrocolloids commonly referred to as "gums" are long-chain, high molecular weight polymers that dissolve or disperse in water to give a thickening or gelling effect and exhibit related secondary functional properties, such as emulsification, stabilisation and encapsulation (Glicksman, 1979). Some functional properties of hydrocolloids that could be applicable for use in coating technology are listed in Table 8 9 and the major hydrocolloids used in food products are listed in Table 9. Table 8. Some functions of gums in food products Function Food application Coating agent Confectionary Emulsifier Salad dressings, soft drinks Encapsulating agent Powdered "fixed" flavours Film-former Sausage casings, protective coatings Protective colloid Flavour emulsion Thickening agent Jams, sauces, gravies (from Glicksman, 1979) 70 Table 9. Classification of major hydrocolloids used in food products Natural Modified natural Synthetic Plant exudates Cellulose derivatives Polyvinylpyrolidene (PVP) Gum arabic Carboxymethlcellulose Polyethylene oxide Gum tragacanth Methylcellulose polymers (polyox) Gum karaya Hydroxyethycellulose Gum ghatti Hydroxypropylmethyl cellulose Seaweed extracts Other derivatives Agar Modified starches Alginates Low-methoxyl pectin Carrageenans Propylene glycolalginate Furcell aran 9 Plant and seed gums Guar Locust bean Cereal gums Starches Plant extracts Pectin Fermented gums X an than Dextrans Animal-derived Gelatin Caseinates (from Glicksman, 1979) 71 The usefulness of most gums is based on their ability to modify the basic properties of water. In food emulsion systems, hydrocolloids are generally used to stabilise the dispersed oil droplets against separation from a continuous phase (generally water). In many applications, the plant hydrocolloids act as emulsifiers. In others they act as thickeners or stabilisers of emulsions which would otherwise separate. The function of plant hydrocolloids in food emulsions is therefore two-fold : 1) they form protective coatings around the particles of dispersed oil, thereby preventing these particles from coalescing and separating from the water phase, and 2) they increase the body or viscosity of the emulsion, thereby causing more resistance against rising oil particles. Gum arabic, being a water-soluble, emulsifying gum, gives emulsion stability to a solution by forming a protective film around each oil particle. Gum tragacanth, unlike gum arabic, is a swelling or suspending gum and depends primarily upon its thickening action to prevent the dispersed oil from coalescing and rising. It is also a bifunctional emulsifier ie increases the aqueous phase viscosity and also lowers the interfacial tension between the oil-in-water emulsion. This eliminates the need to incorporate surface-active agents. 2.10.2.5 Properties and functions of emulsifiers An emulsifier has, firstly, to facilitate the production of a new interface by lowering the interfacial free energy, and secondly, to provide short-term stability by forming a protective adsorbed layer at the oil-water interface. Long-term stability of oil-in-water emulsions may be achieved by thickening the aqueous side of the oil-in-water interface. Most polysaccharides act as stabilisers through their modification of the rheological properties of the aqueous dispersion medium (thickener). Proteins on the other hand act primarily through the properties of their interfacial films, and are therefore both emulsifiers and stabilisers in many instances. An emulsion is most effectively stabilised 72 when a drop of the oil phase just fails to spread on the surface of the emulsifier solution. (Ross et al., 1959). Some important choices have to be made over the volume fraction of oil to be used, the emulsifier concentration and the method of emulsification (Nakamura 1986). Yukagaku (1984) reported that the emulsion stability increased with the molecular weight of the gum arabic stabiliser. This in turn implies that the emulsion stability increases with the film thickness, since larger stabilising molecules might be expected to form thicker layers. 2.10.2.6 Formation of emulsion t The modem theory of the mechanism of surface action is related to the fundamental work of Harkins and Langmuir (1932) dealing with the phenomenum of molecular orientation at interfaces. All substances capable of surface action are composed of relatively large molecules which contain widely separated groups of dissimilar nature. At the boundaries of the solvents there is an orientation of molecules according to the nature of the substances forming the interface. In an oil/water solution, the hydrophobic hydrocarbon chain or "tail" of the molecules orients towards the oil phase. Forces arising from the incompatibility of the hydrocarbon chain results in surface-active molecules or ions migrating to the interface of the solution. The incompatibility seeks to expel the hydrocarbon part of the molecule from the body of the solution. These forces not only produce adsorption at interfaces but also produce aggregates of colloidal dimensions throughout the body of the solution. After an emulsion is formed, the surface-active molecules orientating at the surface of the droplet coalesce upon contact with each other. 73 Certain emulsions do not leave a uniform, continuous film on the surface after dipping. Some collect into a few large drops on the underside of the vegetable, whilst with some others a continuous film forms at first, then breaks a few seconds after dipping to leave a large portion of the surface uncovered by the wax. These are all due to improper wetting of the vegetable surface by the emulsion (Platenius, 1939). 2.10.2.7 Application of emulsion (wettability) Wenzel (1936) observed that the ease with which a solid surface can be wetted' by a liquid depends on three factors : 1) the surface tension of the liquid, 2) the inherent resistance of a given substance to being wetted by a certain liquid and 3) the relative roughness of the surface. It is generally known that lowering the surface tension of an emulsion increases its wetting property. This however does not mean that a liquid of a definite surface tension wets all solid surfaces equally well, nor does it follow that a definite change in surface tension of the emulsion is associated with a corresponding change in the wettability of different surfaces. Wenzel (1936) also called attention to the fact that a given solid surface is wetted more readily if the surface is rough than when smooth because of the increase in surface energy of the solid for a given geometrical area. The wettability of a vegetable may also be influenced by a possible chemical reaction between the emulsion and the epidermal layer of the vegetable. A normal emulsion will completely cover the surface of potatoes, carrots and any non- waxy surface, but spreading agents have to be added when they are used for fruits with waxy skins. Addition of more soap (used as emulsifier) during preparation of emulsions does not improve their spreading power but rather the emulsions become more viscous. It is therefore necessary to add spreading agents to the emulsions after preparation. 74 Trout (1942) advised the use of non-alkaline spreaders. This avoids excess alkali which increases the rate of wilting of fruits and vegetables. 2.10.2.8 Wetting of solids "Wetting agents" are the most common liquid-solid surface active materials. Basically water-soluble compounds orient at the surface with the polar heads of the molecules in the water phase and the non polar tails towards the solid. Some surface-active agents such as phosphatides and oxidised and polymerised glycerides increases the plasticity or viscosity of suspensions by decreasing the adhesional forces between solid particles. A water soluble surfactant adsorbs strongly to an interface towards the air or towards an oil because of its dual structure ie a hydrocarbon tail with insignificant interaction with the water (the hydrophobic part) and a polar group with strong interaction with water (the hydrophilic part). An oil soluble surfactant does not adsorb towards the oil/air interface, but does towards an oil/water interface. 2.10.2.9 Nature of wax film In order to understand the mechanism by which water vapour and other gases diffuse through the wax film it is necessary to consider the thickness of the membrane and the various substances deposited and the manner in which they are arranged in the film. Wax particles in low concentrations of emulsions are recognised as widely spaced, irregular objects, which exhibit rapid Brownian movement as long as sufficient water is present (Platenius 1939). If a wax emulsion is applied in more concentrated form, the wax particles clump together, and at a definite concentration they appear to form a mono-particle layer in 75 which the interspace has been reduced to a minimum. Platenius (1939) concluded that diffusion of water vapour and other gases, depends on their solubility in the film. For example the solubility of water is greater in soap than it is in paraffin. The mechanism of the diffusion of water vapour through the wax film is explained by the dissolving of the water vapour into the continuous soap phase of the film. Due to its continuity, the film soon becomes saturated and allows the water to evaporate from the opposite surface. Platenius (1939) also obtained similar results for the diffusion of other gases such as air and CO2 , where both diffused through the moist wax film in almost direct proportion to their solubility in water. The diffusion rate was reported to be very low when both the membrane and the gases were dry, indicating that the diffusion of any gas depends 9 primarily on its solubility in the continuous soap phase of the film rather than on the porosity of the entire membrane. It was therefore concluded that the relative amount and the type of soap and other dispersing agents used is of greater importance than the type of wax in the emulsion. The moisture retaining properties of wax film could be improved if a soap which absorbs little water is used. The same result might be obtained if dispersing agents, which could possibly reduce the proportion of soap in the emulsion, is used. This, of course, would bring the wax particles closer together, limiting the space occupied by the soap medium which serves as the major path through which the water vapour can escape. A reduction in the rate at which water vapour diffuses through the membrane correspondingly decreases its permeability to oxygen and carbon dioxide. Thus, it is important to prepare emulsions from substances in which these gases dissolve more readily. Other factors such as surface tension and stability of the emulsion is also considered. 76 Postharvest applications of fungicides to control postharvest decay of peaches and nectarine was more effective with the fungicide suspended in a wax emulsion rather than in water (Wells, 1972) Fungicides such as Borax have been incorporated in some of the commercial emulsions for controlling mould wastage due to Penicillium. Excessive alkali in Borax however tends to counteract the beneficial effect of the emulsion in retarding water loss. Non- alkaline fungicides should therefore be considered. 2.10.2.10 Thickness of wax film The thickness of the wax film bears a definite relation to the effectiveness of the film, the t quantity of emulsion used, and the appearance of the vegetable. The relative smoothness of the surface to be coated predominantly determines the actual quantity of material which adheres to the surface after dipping into emulsions of different concentrations. Platenius (1939) reported that the amount of wax on a treated vegetable is exceedingly small. Subsequently, the quantity of wax consumed if waxed vegetables are eaten raw could possibly not be harmful. 77 3 MATERIALS AND METHODS 3.1 Produce Fourteen kinds of fruit and vegetables were studied in this work (Table 10). ‘Hass’ avocado, banana, capsicum, carrot, cucumber, custard apple, lychee, mango, nectarine, plum and zucchini were harvested by their growers; and picked up immediately after arrival at the Flemington wholesale market. Each produce was from the same grower, who determines the commercial maturity of the produce to allow for uniformity in maturity within and between seasons. Commercially matured Granny Smith apples and Packham pears were selectively harvested from Sunbright Orchard, Bathurst NSW and Nashi (Nijisseiki cv) from Nashi Pear Orchard, Shepparton Victoria. Randomisation of the fruits was done in the orchard. As a result of the wide variability between fruits from different trees, it was important that representation of all trees selected was made in each experimental unit. Each experimental unit consisted of 20 fruits. All experimental commodities were carefully selected for uniformity in size, colour and absence of blemishes. Preliminary experiments were carried out over one season with 3 replicates of 10 produce for each treatment, while subsequent experiments were repeated Table 10. Physiological and biochemical properties of produce studies .£ X) .2 13 .2 .b •n •s £ CQ 04 w 1>% jd _o 1 2 S § 1 *43 < O » m •C r2 ■g ■s i 2 *§) — g o o CL, & cd o o * £ 3 c < < I *E 13 T3 •2 t> 2 ; £222 > cd o 3 cd o £ 3 s; i g •& i •c H 2 cd o o o & £ g V-4 I *C u .2 2 *§> 1 2 & o — £ cd o 3 o * H *C 5 u Q 2 * a £ £ c o 3 3 o O 3 3 <3 £ £ 3 3 — o * * • *c J5 u J5 •2 ” — 2 3 g o o o o O «o o c o cd £ O * a 3 3 3 L-, 3 3 a i H a •§> 2 x: *c u J T 2 to 2 > 4> t>0 <3 cd c3 & cd C 3 to 3« 79 H 2 •§b '■§ *c -3 13 if JC <5 <3 •5 § £222 2 o C c £ o a & 3 o 3 3 •§> 2 •& p 6 *3 •5 M I 2 2 O £ o cd o & o bo 5 3 sc 1 i :& • *C 2 2 1 S 55 -3 -§b • 2 £ o £ O 3 o r-H £ cd o i 3 ■ « •§> *C I 2 £ 13 a* *3 £ £ £ o 2 *C 2 •& .§ M X CL -3 t> 2 * . o cd 2 .§ 'C "fj * 2 i* *"§ £ A cd o O 3^ 3 3 SC O £ h > 2 •8 ” H ON Coating materials used for this study includes fats, oils, cellulose gum, starch, emulsifiers and surfactants. Their brand names and sources of supply are listed in Table 11. Table 11. Brand names and sources of supply of coating materials used. Coating materials Brand name/Suppliers 1. Lipids : fats and oils Saturated lipids Butter Allowrie Lard Allowrie Dripping Allowrie Monounsaturated lipids Single fractionation palm olein Malaysian Oil Research Institute Coconut oil Sahib’s choice Peanut oil Chef Olive oil Giralda Canola oil Becol and Topcook Double fractionation palm olein Malaysian Oil Research Institute Polyunsaturated lipids Grape seed oil Borges Safflower oil Crisco Com oil Crisco Avocado oil Castor oil Maize oil Soybean oil Crisco Blended vegetable oil ETA Sunflower oil Crisco and Meadow lea Mineral oil ______Liquid paraffin______B.P Soul Pattinson 80 Table 11 (contd) 2. Cellulose gum Arabic Sigma (USA) Tragacanth Sigma (USA) 3. Starch Sweet potato 4. Emulsifiers Tween 40 Sigma (USA) Tween 60 Sigma (USA) Span 40 Sigma (USA) 5. Surfactant Agral ICI (Australia) 3.3 Preparation and formulation of emulsions Emulsion formulations were developed by screening and testing the compatibility and effectiveness of a number of vegetable oils, cellulose gums, emulsifiers and surfactants. Screening of a wide range of fats and oils was carried out on selected produce (Table 12). Ten of the lipids that gave promising results were then used to coat some commercially important produce (Table 13). Of the 10 lipids, canola, sunflower, coconut and marakot oils were formulated with different concentrations of cellulose gums, emulsifiers and surfactants. Table 14 shows the emulsions made from various combinations and concentrations of these compounds and observations on the stability of the final product The most promising emulsion consisted of 10 parts (by weight) of oil in water. The emulsion was prepared by dissolving lg of tragacanth gum in lOg of sunflower oil (Meadow lea) at ambient temperature. Boiling water (88 mL) was slowly added with 81 Table 12. Lipids trialed in preliminary study The following lipids were trialed1 on capsicum (20°C), cucumber (0° and 20°C), carrot (0° and 20°C), zucchini (7° and 20°C), avocado (20°C) and mango (13° and 20°C). Saturated Monounsaturated Polyunsaturated Mineral oil Butter Single fractionation palm olein Grapeseed oil Liquid paraffin Lard Coconut oil Safflower oil Dripping Peanut oil Com oil Olive oil Avocado oil Canola oil (Becol) Castor oil (Topcook) Maize oil Double fractionation palm olein Soybean oil Blended vegetable oil Sunflower oil (Crisco) (Meadow Lea) 1 Weight loss, colour and firmness were assessed. 82 TJ cs C Table 13. Quality parameters assessed after coating produce with saturated , monounsaturated Vi mineral oils4. Fruit W eight loss % Colour Firmness pH Total Soluble Acidity Physiological Solids disorder Lychee (20°C ) ~7^ ~~P' S i ? 0 CN o o u 'P' -P* 'P' N ectarin e (0° & 20°C ) ~p 83 B an an a i f (20°C ) *> Apple (0° & 20°C ) ~~p ~p ~p ~p ~p Nashi (0° & 20°C ) ~P ~P ' 'P' £ U P h ' ^ B ° Quantities of substances used (g) Oil8 Water Gum Emulsifier Comment Arabic gum 1-5 95-99 6 na unstable 5 5 10-26 na unstable 20 65 15 na unstable 25 50-55 20-25 na unstable 25 45 30 na stable but too thick 25 55 20 na unstable Tween 40 25 55 20 0.55 «! 25 55 20 3 tl 25 55 20 5 ft Span 40 25 45 30 0.45 If 20 80 na 1 II Tween 60 cr It o 40 na 0.8 40 80 na 1.2 II cr If o 120 na 1.6 40 160 na 2.0 O 200 na 22.4 If 10 90 na 0.5 II 10 90 na 1 stable, not too thick 84 Table 14 (contd.) 10 90 Arabic +tragacanth na unstable (1:1) Tragacanth 20 80 1 na unstable 10 89 1 unstable 10 88 1 Agral (lg) stable a Canola, sunflower, coconut or marakot oil was used, na, not added b These emulsions were trialed on avocado, banana, bittermelon, carrot, cucumber, green beans, kiwi, parsnips, pepper (chilli), snow peas, tamarillo, tomatoes and zucchini. Instability in the emulsion was noticeably the formation of oil and water phases. 85 intermittent vigorous stirring. The mixture was homogenised for 2 min with a Bamix hand mixer. One gram of a wetting agent, Agral 600 was then mixed in. This stock emulsion was then diluted to various concentrations with cold tap water when needed. 3.4 Application of coatings on produce 3.4.1 Lipid coatings Each lipid was applied onto the skin of each produce. The coating was then gently f spread uniformly over the entire surface of the produce using gloved hands. The gloved hands were wiped with a clean cloth after each application to ensure a uniform quantity of coating was applied to each produce within a treatment. New gloves were used for each treatment. 3.4.2 Emulsion The stock sunflower oil-water emulsion (10% w/w oil) was further diluted to 0.6, 1.6, 2.5, 3.33 or 5% (w/w) oil with cold water prior to application. The full strength emulsion (10% w/w) was smoothly spread on the produce by hand due to the thickness of the emulsion. Diluted emulsions were applied by dipping produce individually into the solution for 10 s. Coated fruit were placed on wire trays and allowed to dry at ambient temperature before being packed in boxes lined with polyethylene film. Fruit were stored at 20°C and recommended low temperatures. Table 15 summaries the experiments carried out with the emulsions. 86 Table 15. Studies of oil and oil-based coatings on selected produce Produce and storage Treatment Assessment temperature (°C) criteria Cucumber (20°) 6 drops of coconut, Weight loss, colour and sunflower or marakot oil; shrivelling 2.5 or 3.33% (w/w) sunflower emulsion - Avocado (20°C) 0.6, 1.6, 2.5 or 5% (w/w) Weight loss, colour sunflower emulsion respiration rate and internal atmosphere Nashi and Apple 6 drops of coconut, Weight loss, colour (0° and 20°C) sunflower or marakot oil; firmness, pH, acidity, 3.33, 5 and 10% (w/w) TSS, respiration rate, sunflower emulsion internal atmosphere, skin permeability to gases sensory evaluation and physiological disorder. a-famesene and putrescine assessment (apple only) 87 3.5 Produce assessment Produce were analysed for physical and biochemical changes at regular intervals throughout storage. Weight loss, changes in colour, texture and chemical composition were determined. The effects of coatings on produce respiration, internal O2, CO2 and C2H4 and skin resistance to gaseous exchange were examined. The development of physiological disorders in apple, Nashi and Packham pears were also assessed. 3.5.1 Physico-chemical Analysis t 3.5.1.1 Weight loss Produce were weighed before and immediately after application of coatings. Samples from each treatment unit were then reweighed at regular intervals throughout the storage period. Percentage weight loss was calculated using the following formula : (initial weight - final weight) x 100 initial weight 3.5.1.2 Colour assessment For most fruit, colour was visually assessed on a 1 - 5 scale. For apple, pear, Nashi and cucumber : 1 = green, 2 = 25% yellow, 3 = 50% yellow, 4 = 75% yellow and 5 = 100% yellow. For Santa rosa plum : 1 = green, 2 = <50% red, 3 = 50 - 75% red, 4 = red and 5 = dark red. For lychee : 1 = red, 2 = dark red, 3 = slightly reddish brown, 4 = brown and 5 = dark brown. For Hass avocado : 5 = green, 4 = 25% purple, 3 = 25 -50% purple, 2 = 50 - 75% purple and 1 = purple/black. The colour of banana was assessed using a standard banana ripening chart (United Fruit Sales Corp. Boston, Mass, USA) with 88 colour plates ranging from 1 to 7 where 1 = green, 4 = more yellow than green and 7 = yellow flecked with brown. The skin colour of apples, in some experiments, was measured with a Minolta chroma meter (model CR300, Osaka) after standardising the instrument with a standard white plate tile (calibration plate CR - A43) at ambient temperature. Each fruit was held on the palm and colour readings taken at 5 random points on the fruit surface. Colour values were recorded as the "a" value (greenness). 3.5.1.3 Measurement of firmness Fruit firmness was measured with an Effegi hand penetrometer (Effegi, 48011, r Alfonsine, Italy) with a 11mm plunger. A thin slice of fruit peel was first removed with a sharp knife from the site of pressure testing. The firmness of the fruit was expressed as the force required to press the plunger into the flesh (kg). Measurements were made at 4 points around the equatorial region of each fruit. Five fruits were used from each unit Avocado firmness was subjectively assessed by placing the fruit on the palm and exerting a slight pressure. A scale of 1 - 5 was used with 1 = very hard, 2 = firm, 3 = slightly soft, 4 = soft and 5 = very soft. 3.5.1.4 Measurement of pH The pH of the undiluted fruit juice was measured at 20°C using a digital pH meter (T.P.S., Brisbane) equipped with a combination of electrode and automatic temperature compensator. 89 3.5.1.5 Titratable acidity Titratable acidity in fruit was assessed using the AOAC method (942.15, 1980). A fruit juice aliquot (25g) was quantitatively transferred to a beaker containing lOOmL distilled water. The solution was constantly stirred using a magnetic stirrer and the initial pH measured by a pH probe suspended in the solution. The solution was titrated with 0.1N Sodium hydroxide (Ajax, Sydney) to a pH of 8.2. The end point was taken when the pH remained at 8.2 for 30 secs. The titratable acidity was calculated as citric (pear, plum and nectarine) and malic (Nashi and pear) acid equivalents. Titratable acid = 0.1 x 100 x acid equivalent x titre value (NaOH used) (milliequivalent/lOOg) ' 25 3.5.1.6 Total soluble solids The total soluble solids of the juice was determined at 20°C using a bench top refractometer (Bellingham and Stanley, London) and the values expressed as °Brix. 3.5.1.7 Sensory evaluation Sensory evaluation was carried out with 5 untrained assessors. Nashi fruits were assessed after 33 days storage at 20°C and apples after 20 weeks at 0° (and 7 days at 20°C). Whole fruits were peeled and sliced by hand into 1-cm thick vertical slices, making certain the core was excluded. One slice from each treatment was placed on separate shallow dishes labelled with a 3-digit random number. Sample preparation was done quickly to minimise enzymic discolouration of the fruit slices prior to assessment. Assessors evaluated the samples under normal laboratory conditions. The qualities evaluated were texture, flavour and overall acceptability. Overall acceptability was 90 scored after considering the texture and flavour, using a 9-point hedonic scale. Sensory evaluation was reported as the average of the scores of the assessors. The following scaling system was used for texture, flavour and overall acceptability : Texture Acceptability : 1 to 9 5 = very crispy 1 = dislike extremely 4 = moderate crispy 2 = dislike very much 3 = slightly crispy 3 = dislike moderately 2 = less crispy 4 = dislike slightly 1 = soft 5 = neither like or dislike « 6 = like slightly 7 = like moderately Off-flavour 8 = like very much 1 (none) - 7 (strong) 9 = like extremely 3.5.1.8 Assessment of Physiological disorders Physiological disorders such as superficial scald, core and flesh browning occur in fruits such as apple, pear and Nashi after long-term cold storage. Studies were carried out to investigate the possibility of using oil and oil emulsion coatings to control the development of physiological disorders in apple, pear and Nashi stored at 0°C. Experimental units were set up as in Sections 3.1 and 3.4. Additional untreated units of 6 x 20 fruit were obtained from the same randomisation as the experimental units and were stored as pilots with the experimental units at 0°C. A pilot unit was removed from storage each month and held at 20°C for 7 days to monitor the development of physiological disorders. When a high proportion of the pilot fruit had developed typical 91 symptoms, all corresponding experimental fruit were removed from storage and held at 20°C for 7 days to allow the disorders to develop fully. Fruits were assessed externally for superficial scald and then cut into transverse slices for internal examination for core and flesh browning. Severity of physiological disorders such as superficial scald, core and flesh browning was subjectively scored using a 1 - 5 score scale, where 1 = no disorder, 2 = slight, 3 = moderate, 4 = severe and 5 = very severe disorder. Studies were also undertaken to develop an understanding of the fundamental effect of coatings on the development of the physiological disorders. This was done by analysing the coated and uncoated fruit for a-famesene and putrescine as discussed below. 3.6 Extraction and analysis of cc-farnesene Famesene was analysed using the method of Meigh and Filmer (1969). Samples of 5 fruit were weighed and the circumference of each fruit measured, first in the direction passing through the stalk and calyx and then in the direction at right angles to this. Each apple was fixed on a spike, rotated for 30 s in a beaker of ether (A.R. quality, lOOmL; Ajax, Sydney) and rinsed with 5mL fresh ether. Each batch of lOOmL of ether was used for 5 fruits only. The ether washing containing the apple wax were bulked and dried with sodium sulphate. The ether was evaporated under vacuum and the wax was rapidly transferred to 20mL light petroleum (A.R quality b.p 30 - 40°C, AJAX, Australia). The mixture was filtered through glass paper (Whatmann Maidstone, England) to remove the insoluble ursonic acid, concentrated to lOmL under vacuum and fractionated on an alumina column (100 - 200 mesh,12cm x 12mm ID; Unilab, Ajax, Australia). The hydrocarbon fraction was eluted in light petroleum (150mL), 92 concentrated and then made up to lOmL. The homogenates were taken up in 2.5cm^ syringes and passed through Swynex filter holders fitted with Whatmann GF/B glass micro-fibre filters, into collecting vials. One microlitre of aliquot was injected into the column of a GC (Varian series 3300) with a 10|iL microsyringe (Hamilton Co.). The separation was carried out in a capillary column packed with BP 20 (polyethylene glycol, 12m long, 0.221mm ID). Nitrogen was used as the carrier gas, with a flame ionisation detector. Gas flow rates were nitrogen 2mL/min, hydrogen 30mLVmin and air 300mL/min.' The column temperature was 115°C, and the injector and detector temperatures were 150°C. Famesene extracted from apples immediately after harvest was used as a standard. The ___ r same procedure as above was carried out. The collecting vial was however weighed before collecting the homogenate. The vial containing the extract was held under nitrogen gas until the solvent was evaporated. The vial was then re weighed. The difference in weight was taking as the amount of famesene in the standard. The response peak at a particular concentration was used to identify and calculate the a- famesene in the sample and quantified as pg/lOOcm^ surface of fruit. cx-famesene = sample peak area x concentration of standard standard pk. area 3.7 Extraction and analysis of Putrescine Putrescine was extracted from the apple skin using the method of McDonald and Kushad (1986). Five grams of peel was collected from 5 fruit and blended for lmin at 4°C in 50mL of 5% (v/v) HCIO4 (AJAX, Australia) to give a final concentration of O.lg/mL, using an Omnimixer (Omni, USA). The homogenised mixture was centrifuged (Sorvall, 93 Germany) at 3000rpm for lOmin and the supernatant used for the analysis of putrescine, according to the procedure of Redmond and Tseng (1979). Two millilitre of the tissue extract was mixed with 2mL of 4N sodium hydroxide and 5pL benzoyl chloride (May and Baker, England). The mixture was vortexed (Extex, Australia) for 15 secs and incubated for 20 mins at 35°C in a waterbath. The reaction was terminated by the addition of 4mL saturated NaCl and the benzoylated putrescine was extracted with 2mL chilled diethyl ether. The ether fraction was collected and centrifuged at 3000rpm for 5 mins. Two millilitres from the organic phase was taken and evaporated in a nitrogen stream. The residue was then resuspended in 200pL methanol, and a 5pL aliquot was injected into a Waters HPLC 9 (Model 441) equipped with a UV detector set at 254nm and a C-18 reverse phase column (25cm, RP-8, 10pm Bondapak). The mobile phase was methanol/water (58:42) at a flow rate of 1.5mL/min. Standard putrescine (O.lmL, Sigma, USA) was passed through the same procedure as above. Putrescine from the samples was identified by comparison with the retention time of the putrescine standard and quantified as pg/g of peel. putrescine = sample peak area x concentration of standard standard pk. area 94 3.8 Physiological Measurements 3.8.1 Respiration rate The respiratory behaviour of uncoated and coated fruit was investigated to determine the effect of the coating on respiration. Respiration was determined as CO2 evolved per hr and was measured at regular intervals of 24 h. Pre-weighed fruit was placed in a 500mL container with a continuous stream of air passing through the container. The flow rate of the air passing over the fruit was maintained at approximately 1.4L/hr at 20°C by means of needle valves downstream of the respiration vessels. Location of the incoming and outgoing lines at the bottom and 9 top, respectively, of each container prevented accumulation of CO2 and ensured good circulation of air around the fruit. The outlet tubes from the containers were connected to an automatic sampler (Analgas, Australia) connected to an Infra Red Gas Analyser (Model PIR-2000, Horiba, Japan). The infra-red flow cell was connected to a recorder whose displacement was representative of the concentration of CO2. The analytical cell was calibrated daily with a standard gas mixture of 0.652 ± 0.013% CO2 in air (p standard, CIG, Sydney) fed into the cell from a gas cylinder. The respiration rate was calculated as described and expressed as mL CO2 kg"l.hr'l. sample reading x standard C02(%) x flow rate (L/hr) standard reading fruit weight (kg) 3.8.2 Internal atmosphere Several workers have used vacuum extraction for obtaining samples of gases from inside fruits and vegetables. Brooks (1937) described a method involving a Toricellian vacuum produced by raising and lowering a mercury column. Beyer and Morgan (1970) used a 95 vacuum pump for studying the ethylene content of plant tissues, a method essentially similar to that employed by Maxie et al. (1965) in their study of the internal atmosphere of lemons. This method was also employed in this study. A 20-litre Plexiglas vacuum chamber was filled to 90% capacity with a degassed saturated solution of ammonium sulphate (solubility of 76.7g/100g cold water). A salt solution is used in preference to water since solubilities of gases decrease with alkalinity or acidity. Degassing of the solution was done by exposure to vacuum (about lOOmmHg) for several hours before use. A collection flask (an inverted filter funnel plugged with a rubber septum) was completely immersed in the degassed solution and air bubbles adhering to the inner surface of the flask and the rubber septum cap were removed. Fruit to be analysed were immersed in a 0.01% solution of Tween 20 (Sigma, USA) to reduce adhesion of air bubbles and immediately placed underneath the collection flask by slighdy tilting it. The vacuum chamber was sealed and a partial vacuum of lOOmmHg applied for 2 min. Gas bubbling out of the fruit rose to the top of the inverted flask and collected under the rubber septum. The vacuum was released and 15 1-mL gas samples were collected with a syringe. Plate 1 shows the experimental setup. 96 Plate 1. Apparatus used for vacuum extraction of the internal gases. 97 3.8.3 Measurement of CO2 and O2 Samples of gas were injected into a gas chromatograpi (Model Series 350, GOW-MAC, Bridgewater, N.J USA) equipped with a Porapak Q (180mm length) and molecular sieve (typ>e 5A, 30 - 60 mesh, 90mm length) columns, operating at a column temperature of 60° The instrument was standardised with a gas mixture containing 1.0 ± 0.22% CO2, and 2.03’> ± 0.04% O2 (CIG, Sydney). The analysis vas run with a Maxima program (Manama 820, version 3.3, Millipore Corp.). Calculaton of the concentrations of gases was done using the chromatographic peak area data. Values for samples were obtained using peak areas corresponding to the retention time fcr standard CO2 and O2. CO2 (%) = peak area of sample x 1.0 peak area of standard O2 (%) = peak area of sample x 2.03 - 1.04 peak area of standard The factor 1.04 was used to correct for argon which is eluted with oxygen. 3.8.4 Measurement of ethylene Gas was injected into a Gas Chromatography (Model 1400 Varian Aerograph, Palo Alto, California) equipped with a flame ionisation detector and stainless steel column (80 - 100 mesh., 60cm x 2.5mm internal diameter) packed with Porapak Q (Supelco, Bellefonte, PA). It was operated at column temperature of 50°C, injector and detector temperature 135°C. Nitrogen, hydrogen and air flow rates were 50, 40 and 300mIVniin, respectively. The instrument was standardised with a gas mixture containing 2.7fiL/L ethylene in air. 98 The concentration of ethylene in the samples was determined against the peak height of ethylene in a standard gas mixture containing 2.7±1 pIVL ethylene in air/nitrogen (p-std, CIG, Sydney). Ethylene concentration (|iL/L) was calculated as : sample peak height x sample attenuation ______x standard concentration standard peak height x standard attenuation 3.9 Determination of skin permeability Determination of the fruit skin permeability to oxygen and carbon dioxide was carried out using the method of Banks (1984). The permeability of the fruit surface to each gas was estimated using the rate of release or uptake of that gas by the fruit and its gradient across the fruit surface. The surface area (A) of each fruit was calculated using diameter readings taking horizontally and vertically. Individual fruits were weighed and placed in sealed 8 litre (V) containers for lhr. The gaseous contents of the atmosphere in each container were measured before and after the fruit had been in the container for 1 hr. The difference in the initial and final gas compositions gives the changes in concentration of gases in the container (R). Internal atmosphere composition of the fruit was determined immediately after removal from the container using the method described in Section 3.7.2. Samples were then analysed for CO2, and O2 as in Section 3.7.3. The skin permeability of fruit to the each gas was estimated using the following equation: 99 P = R x (V-Wl 100 x A x t x AC where P = cm h' 1 mbar" 1 R = change in concentration of each gas in the container (%) V = volume of container (mL) W = volume displaced by fruit in water (mL) A = surface area of fruit (cm^) t = time over which fruit was sealed (hr) AC = gradient in concentration across fruit skin (mbar, estimated as the difference between the internal composition and the mean of the initial and final composition of each gas in the container). 3.10 Electron microscopy of coatings Coated and uncoated fruit were stored at 20 °C and sampled at 1 week intervals for microscopic study. Portions of peel (lcm^) of 1mm thickness were plunged into liquid nitrogen at -196°C and then freeze dried for 24 hr in Balzers BAF 400 freeze fraction system. The dried samples were coated with gold under the Polaron Sputter Coater. This was to improve the conductivity of the samples. The samples were observed under Cambridge Stereoscan S-360 using a backscatter imaging system.. 3.11 Statistical analysis Analysis of variance was used to determine the treatment effects in this work. The variance ratio, or F-statistic relates the variance due to treatment (MSy) with the 100 residual variance (MSg or error mean square). The precision with which treatment effects are estimated is dependent on the accuracy of estimation of the error mean square which represents the random variation between results from individual experimental units. A wide variability occurs between fruit from different trees. It was important that representation of all the trees selected was made in each replicate of the treatments (this was possible only for fruits harvested personally). A randomised block design was used in which experimental materials were divided into "blocks". Experimental units within a block were more similar to each other than they were to those of other blocks. In this case each unit contained a similar number of fruit from each tree harvested by each picker. Each of the treatments was represented in the same number of experimental units (2) in each block. A block therefore represented one complete replicate of the experimental treatments. Each treatment unit contained 20 fruit replicated twice for each picking/harvest. Data was analysed by analysis of variance using the General Linear Model (GLM) procedure, a package program of The Statistical Analysis System (SAS Institute, Cary, NC USA). Means separation were carried out using Duncan’s Multiple Range Test (DMRT) at p = 0.05. 101 4 RESULTS 4.1 Preliminary studies The preliminary studies were conducted to attain an understanding of the potential effects of lipid-based skin coatings on the postharvest behaviour of some commercially important fruit and vegetables. A range of fruits and vegetables were coated with skin coatings made from selected fats and oils with different properties, especially degree of saturation, to determine their physical, chemical and physiological responses to each coating during storage at various temperature. r 4.1.1 Non-climacteric produce 4.1.1.1 Capsicum In this study, green capsicums coated with 21 different lipid coatings were stored at 7° and 20°C and assessed for the development of red colour and shrivelling. Uncoated capsicums at 20°C started to degreen and developed red colour after 5 days of storage and were fully red after 8 days (Table 16). In contrast, capsicums coated with castor, peanut or avocado oils retained full green colour until day 15, while those coated with canola, sunflower (Crisco), double fractionated palm olein, liquid paraffin, soybean, maize, olive or blended vegetable oils did not begin to degreen until after 18 days of storage (Table 16). Not all coated capsicums attained full red colour during stored at 20°C (Table 16). Capsicums coated with safflower or grapeseed oils became fully red after 18 days while 102 Table 16. Effect of lipid coatings on green colour retention of capsicum at 20°C Treatment Days to appearance of red colour Days to full red colour Control (untreated) 5a 8a Saturated lipids Butter 8a - Lard 9a - Dripping llab - Monounsaturated lipids Single fractionation palm olein 9a - Coconut oil llab - Peanut oil 15cd 21c Olive oil 18d - Canola oil (Becol) 18d - (Topcook) 16cd - Double fractionation palm olein 18d - Polyunsaturated lipids Grapeseed oil 9a 18b Safflower oil llab 18b Com oil 13bc - Avocado oil 15cd - Castor oil 15cd 21c Maize oil 18d - Soybean oil 18d 28 d Blended vegetable oil 18d - Sunflower oil (Crisco) 18d 28d Sunflower (Meadow Lea) 18d 28d Mineral oil Liquid paraffin 18d - Six drops of oil (about 20 mg) or approx. 25 mg of butter, dripping and lard were smoothly spread on each capsicum. Each value is the mean of 3 replicates of 10 capsicums. Values followed by different letters within columns are significantly different from each other at p = 0.05 by Duncan Multiple Range Test. (-) Experiment was terminated before full red colour developed due to excessive shrivelling. 103 those coated with peanut or castor oil attained full red colour after 21 days. The most effective coatings in delaying the development of red colour were soybean and sunflower oils. Capsicums treated with soybean or sunflower oil only completely degreened after 28 days storage. Capsicums treated with butter, lard, dripping, SFPO, coconut, olive, canola, DFPO, com, avocado, maize, blended vegetable oils or liquid paraffin failed to completely degreen before they became unmarketable due to severe shrivelling. At 7°C both coated and uncoated capsicums remained green after 34 days of storage. The uncoated capsicums were still acceptable for marketing but the experiment was terminated due to mould development on the coated capsicums. Shrivelling was the major determinant of the marketable life of coated as well as uncoated capsicums. Since both green and red capsicums are acceptable commercially, the storage life of capsicum in this study was assessed as days to a moderate level of shrivelling (score 3). Shrivelling in uncoated capsicums was moderate after 10 days of storage at 20°C (Table 17). Shrivelling was however, significantly delayed (p = 0.05) by some of the coatings. Capsicums coated with castor, Single Fractionated Palm Olein (SFPO), blended vegetable oil or safflower oils were moderately shrivelled after 18 days and 21 - 28 days in those coated with lard, dripping or coconut oil. Butter, liquid paraffin, as well as peanut, olive, DFPO, canola, avocado, com, grapeseed, maize, soybean, and sunflower oils, however, had no effect on delaying shrivelling of capsicums at 20°C. 104 Table 17. Effect of lipid coatings on shrivelling of capsicum at 20°C Treatment Days to moderate shrivelling Control (untreated) 10a Saturated lipids Butter 11a Lard 21bc Dripping 28bc Monounsaturated lipids Peanut oil 10a Olive oil 10a Double fractionation palm olein 10a Canola oil (Becol) 11a (Topcook) 11a Single fractionation palm olein 18b Coconut oil 28bc Polyunsaturated lipids Avocado oil 10a Com oil 11a Grapeseed oil 11a Maize oil 11a Soybean oil 11a Sunflower oil (Crisco) 11a Sunflower (Meadow Lea) 11a Blended vegetable oil 18b Castor oil 18b Safflower oil 18b Mineral oil Liquid paraffin 11a Six drops of oil (about 20 mg) or approx. 25mg of butter, dripping and lard were smoothly spread on each capsicum. Shrivelling was assessed as 1 = none, 2 = slight, 3 = moderate and 4 = severe. Each value is the mean of 3 replicates of 10 capsicums. Values followed by different letters are significantly different from each other at p = 0.05 by DMRT. 105 At 7°C, uncoated capsicums remained unshrivelled while slight shrivelling was noticed on capsicums treated with canola, safflower, soybean peanut or blended vegetable oils before the experiment was terminated after 34 days due to mould development on the coated capsicums. Overall, lipid coatings appeared to have significant potential for extending the shelf life of capsicum at 20°C. However, although dripping, lard, coconut, castor, safflower, SFPO and blended vegetable oils were highly effective in extending the storage life of capsicums, capsicums coated with dripping, lard and SFPO were sticky to touch, while t castor oil produced excessive glossiness not natural to capsicums. 4.1.1.2 Cucumber In this study, green cucumbers treated with 21 different lipid coatings were stored at 7° and 20°C and assessed for the development of yellow colour and shrivelling. As the marketable life of cucumber is considered to be inversely related to the appearance of yellow colour, storage life of cucumber was assessed as the time taken to the appearance of unacceptable yellow colour (score 3: 10 - 25% yellow). Uncoated cucumbers started to yellow after 7 days storage at 20°C. Except for grapeseed oil, all coated cucumbers retained a full green colour significantly (p = 0.05) longer than the uncoated (Table 18). Cucumbers coated with coconut, sunflower, liquid 106 paraffin, olive, maize or safflower oils, as well as dripping remained fully green for more than 17 days of storage at 20°C, which was about 9-13 days longer than the uncoated cucumbers (Table 18). Avocado and castor oils were the most effective, retaining the green colour of cucumbers for 22 days longer than the uncoated cucumbers. Uncoated cucumbers developed unacceptable yellow colour after 13 days storage at 20°C. Application of coating significantly (p = 0.05) extended the storage life of cucumber with coated cucumbers developing unacceptable colour after 23-41 days (Table 18). Grapeseed, sunflower (Meadow Lea), Single Fractionated Palm Olein, 9 avocado, castor and safflower oils were the most effective, extending the storage life of cucumbers by about 3 weeks (Table 18). Coated and uncoated cucumbers stored at 7°C remained green after 25 days of storage but the experiment was terminated at day 25 due to development of tissue breakdown in the form of soft patches on most of the coated cucumbers. In addition to tissue breakdown, mould was also observed on cucumbers treated with maize, soybean or grapeseed oils. 107 Table 18. Effect of coatings on green colour retention of cucumbers stored at 20°C Treatment Days to appearance of Days to unacceptable yellow colour yellowing* Control (untreated) 7a 13a Saturated lipids Butter 16c 23b Lard 16c 26bc Dripping 18cd 29bc Monounsaturated lipids Canola oil (Becol) 16c 22b Peanut oil 16c 26bc Coconut oil 18cd 29bc Double fractionation palm olein 16c 29bc Olive oil 20cd , 31bc Canola (Topcook) 16c 31bc Single fractionation palm olein 16c 39c Polyunsaturated lipids Com oil 13bc 23b Soybean oil 13bc 23b Blended vegetable oil 16c 23b Sunflower oil (Crisco) 16c 26bc Maize oil 20cd 6bc Safflower oil 20cd 41d Grapeseed oil lOab 41d Sunflower (Meadow Lea) 20cd 41d Castor oil 29e 43d Avocado oil 29e 43d Mineral oil Liquid paraffin 18cd 23b Six drops of oil (about 20 mg) or approx. 25mg of butter, dripping and lard were smoothly spread on each cucumber. Each value is the mean of 3 replicates of 10 cucumbers. Values followed by different letters within columns are significantly different from each other at p = 0.05 by DMRT. * Unacceptable yellow colour: score 3 (10 - 25% yellow). 108 In general, grapeseed, sunflower and safflower oils appear to have significant potential for extending the shelf life of cucumbers at 20°C. Cucumbers coated with castor oil were too glossy, while avocado oil left a tainted odour on the cucumbers. Cucumbers coated with SFPO were also sticky to touch. 4.1.1.3 Carrot Carrots treated with 21 different lipid coatings were stored at 0° and 20°C. Carrots were assessed for weight loss and shrivelling during storage. Shrivelling was the major problem and storage time was assessed as the time for carrot to reach minimum acceptable shrivelling (score 3: moderately shrivelled). Weight loss of uncoated carrots drastically increased from 1.1% after 1 day of storage to 30.02% after 10 days of storage at 20°C (Table 19). Coating the produce had a marked (p=0.05) effect on reducing weight loss. The most effective coatings in reducing weight loss were butter, lard, Double Fractionated Palm Olein (DFPO) and dripping, reducing weight loss to an average 10.1% in coated carrots. Uncoated carrots started to shrivel after 6 days of storage at 20°C and became unacceptable after 9 days (Table 19). Shrivelling was delayed by all coatings and did not appear on carrots coated with the saturated lipids, as well as those coated with SFPO, coconut or olive oils, until after 29 days. 109 Table 19. Effect of coatings on storage life of carrot stored at 20° and 0°C Treatment Weight loss1 Days to minimum acceptable shrivelling % 20°C 0°C Control (untreated) 30.02a 9a 25a Saturated lipids Butter 11.1c 35d 49c Dripping 12.2cd 35d 49c Lard 7.99b 42d 49c Monounsaturated lipids Canola oil (Topcook) 14.3def 11a 31b Canola oil (Becol) 18.1 fgh 11a 30b Peanut oil 18.82gh 11a 36b Olive oil 14.3def 23c 36b Coconut oil 12.95cd 35d 49c Single fractionated palm olein 12.9cd 35dc 49c Double fractionated palm olein 9.2bc 35dc 49c Polyunsaturated lipids Soybean oil 21.5h 9a 28ab Com oil 19.0gh 11a 28ab Sunflower (Crisco) 18.0fgh 11a 36b Sunflower (Meadow' Lea) 22.5h 11a 36b Maize oil 15.6dfg 11a 28ab Grapeseed 21 4h 15ab 31b Safflower 14.9def 21bc 36b Blended vegetable oil 13.5d 21bc 36b Avocado 12.5cd 26c 31b Castor oil 17.8efgh 26c 31b Mineral oil Liquid paraffin 17.6efgh 13a 36b Six drops of oil (about 20 mg) or approx. 25mg of butter, dripping and lard were smoothly spread on each carrot. 1 Weight loss of carrot after 10 days of storage at 20°C. Shrivelling score: 1 = none. 2 = slight. 3 = moderate. 4 = severe. Minimum acceptable shrivelling: score 3. Each value is the mean of 3 replicates of 10 carrots. Values followed by different letters within column are significantly different at p = 0.05 by DMRT. 110 These lipids were the most effective coatings, significantly (p = 0.05) extending the storage life of carrots, with the coated carrots being moderately shrivelled after 35-42 days of storage, compared to between 11-26 days in the other coated carrots (Table 19). At 0°C, shrivelling in the uncoated carrot became unacceptable after 25 days of storage (Table 19). Coating delayed shrivelling in carrots, with butter, dripping, lard, coconut, SFPO and DFPO being more effective. Carrots coated with these coatings had acceptable appearance with no internal damage or bitter taste until 49 days when they started to develop uneven colour. Overall, SFPO and DFPO, as well as coconut oil are more promising coatings for the storage of carrot, since carrots coated with butter, dripping and lard were sticky to touch. 4.1.1.4 Zucchini Zucchini treated with 21 different lipids were stored at 20° and 7°C and assessed for appearance. Storage life was assessed as time to reach minimum marketable quality based on appearance. At 20°C, uncoated zucchinis started to shrivel after 7 days of storage and were no longer acceptable by day 9 (Table 20). The uncoated zucchinis were soft, as well as being ill severely shrivelled. Not all coatings had a desirable effect on zucchini. Zucchinis coated with lard or avocado remained acceptable for about 11 days. The most effective of the coatings were dripping and castor oil, with the coated zucchinis remaining acceptable after 13 days of storage at 20°C. All other coatings accelerated spoilage in zucchini, with coated zucchinis displaying tissue breakdown, in the form of exudates, within 5 days of storage. This led to rot, accompanied by an offensive odour. At 7°C, uncoated zucchinis and those coated with dripping or coconut oil retained a marketable quality for 28 days of storage (Table 20). All other coated zucchinis had a shorter storage life with the coatings accelerating deterioration in zucchini. Butter, soybean, peanut, canola, SFPO and blended vegetable oils were the least effective, shortening the storage life of coated zucchinis to 14 days. In general, only dripping and castor oil had a beneficial effect (p = 0.05) on the storage life of zucchini stored at 20°C but not at 7°C. Zucchinis coated with dripping were, however, sticky while zucchinis coated with castor were excessively glossy. Lipids are therefore, not promising in the extension of the storage life of zucchini at either 7° or 20°C. 112 Table 20. Effect of coatings on storage life of zucchinis stored at 20° and 7°C Treatment Days to minimum marketable quality 20°C 7°C Control (untreated) 9a 28c Saturated lipids Butter 5a 25c Dripping 13c 28c Lard lObc 27c Monounsaturated lipids Canola oil (Topcook) 5a 14a Canola oil (Becol) 5a 14a Peanut oil 5a 14a Olive oil 5a 20b 9 Coconut oil 5a 28c Single fractionated palm olein 5a 25c Double fractionated palm olein 5a 20b Polyunsaturated lipids Soybean oil 5a 14a Com oil 5a 18b Sunflower (Crisco) 5a 18b Sunflower (Meadow Lea) 5a 18b Maize oil 5a 27c Grapeseed 5a 18b Safflower 5a 18b Blended vegetable oil 5a 14a Avocado llbc 27c Castor oil 13c 25c Mineral oil Liquid paraffin 5a 20b Six drops of oil or approx. 25mg of butter, dripping and lard were smoothly spread on each zucchini. Each value is the mean of 3 replicates of 10 zucchinis. Values followed by different letters within each column are significantly different at p = 0.05 by DMRT. 113 4.1.2 Climacteric produce 4.1.2.1 Avocado Green avocados treated with 21 different lipids (Table 12 - Section 3.3 pg 82) were stored at 20°C and assessed for colour change and loss of firmness. Uncoated avocados were at the eating stage (soft with brown/purple colour) after 5 days storage at 20°C. Coated fruits, however, remained firm and green after 10 days. The experiment could not be continued due to development of mould and stem end rot on all the coated fruits. Fruits were also discoloured internally with blackened flesh. The ripening problem encountered in this experiment could have been due to excessive amount of coatings used on each fruit which was about 30 mg/fruit. 4.1.2.2 Custard apple Green custard apples treated with 21 different lipids (Table 12 - Section 3.3) were stored at 20°C and assessed for colour change and loss of firmness Uncoated fruit were at the eating stage (very dark brown and extremely soft) after 6 days of storage. All coatings allowed change of colour from green to brown, although coated fruits remained firm. Coated fruits except for those treated with lard, butter, dripping and SFPO, remained firm up until day 10 when the experiment was terminated due to rot in the coated fruits. Although custard apples coated with lard, butter, dripping and SFPO were slightly soft, fruits had uneven pulp softness when cut open. Discolouration of the flesh was also observed in all coated custard apples, which is likely to be due to the effect of the coatings on the normal metabolism of the fruit. Due to the physical structure of custard apple, the lipids were brushed onto the fruit and enough was applied to cover the entire fruit. This did not allow for uniform quantities of lipids on the fruit and was often observed to be excessive on some fruits, possibly preventing ripening. 4.1.2.3 Mango Mangoes treated with 21 different lipids (Table 12 - Section 3.3) were stored at 13° and i 20°C and assessed for the development of yellow colour and softening. At 20°C, uncoated mangoes started to develop a yellow colour (score 2: <25% yellow) after 5 days of storage and were fully yellow (score 5) after 10 days. In contrast, the coatings effectively inhibited loss of green colour. Mangoes coated with castor, lard, butter, coconut, olive, blended vegetable oil, canola, peanut or liquid paraffin did not start to degreen until after 10 days of storage, while those coated with com, DFPO, sunflower, canola, avocado, safflower or soybean oil retained a full green colour until day 12. The most effective coatings were dripping and grapeseed oil, with coated mangoes remaining fully green after 18 days. Coated fruits however, did not attain full yellow colour. Mangoes coated with lard, SFPO, avocado or blended vegetable oils were at colour score 4 (75% yellow, 25% green) by the end of the 24 days storage, while all other coated mangoes still retained 50% of the initial green colour. 115 At 13°C, uncoated mangoes started to develop a yellow colour after 9 days of storage and were fully yellow (score 5: 100% yellow) after 18 days. Only fruits coated with lard, dripping, butter, SFPO, sunflower, grapeseed, canola, soybean, peanut, maize or blended vegetable oils attained a colour score of 4 (75% yellow, 25% green) within 26 - 37 days storage. Mangoes coated with castor, com, coconut, DFPO, olive, avocado, safflower and liquid paraffin remained at colour score 3 (50% yellow, 50% green). Uncoated mangoes started to soften after 7 days storage at 20°C and were very soft (score 4) by day 18. Coating markedly retained firmness with most coated fruit starting f to soften after 12 days storage. All coated mangoes except for those coated with castor, lard, SFPO, blended vegetable oil, canola and avocado remained slightly soft (score 2) until the experiment was terminated due to severe shrivelling on the coated fruits after 24 days of storage. Mangoes coated with castor, lard, SFPO, blended vegetable oil, canola or avocado attained moderate softness (score 3) after 18 days and remained so until the end of storage. All coated mangoes however, had a sour taste and fermented odour at the end of the experiment. At 13°C, softening of uncoated mangoes started after 18 days with fruits becoming moderately soft (score 3) after 33 days of storage. Coated fruits on the other hand, were just slightly soft (score 2) after 37 days when the experiment was terminated due to severe shrivelling and development of skin blemishes on coated mangoes. 116 4.1.3 Summary A number of commercially available edible fats and oils were tested on a number of selected produce with different physical and physiological characteristics. In general, most of the produce responded well, but in varying degrees, to the lipid coatings with respect to senescence changes such as softening and colour change. The major problems associated with the application of lipid coatings on fruit and vegetables were greasiness of some coatings whilst anaerobiosis, uneven ripening and tissue breakdown occurred especially with the climacteric produce. Since most climacteric produce start their ripening process during storage, it is important r that the process is not completely hindered. With these preliminary experiments, it was observed that the coatings used, although delaying colour and softness in avocado, custard apple and mango, did not allow for proper ripening of the fruits. The ripening problems may have been due to excessive amount of lipids applied on the fruits. Further work could be done to determine the required quantity of lipid that will allow proper ripening, as well as the application technique to be employed for each of the lipids. The effect of reduced concentration of a selection of lipids was assessed by further experimental work with some produce. 117 4.2 Effect of lipid coatings on selected produce 4.2.1 Introduction As a number of coating gave similar results it was decided that the coating formulation work should be conducted on fewer oils. Oils considered likely to be highly effective and which are representative of various classification of lipids were then selected. Lipids selected for more detailed studies were butter (BT) - milkfat; dripping (DR) - animal fat; coconut (CO) - lauric; peanut (PN) and sunflower (SN) - oleic-linoleic; canola (CA) - erucic; soybean (SB) - linolenic; castor (CS) - hydroxy; liquid paraffin (LQ) - mineral; and marakot (MKT) - a combination of soybean and palm olein. These $ lipids are typical of the different basic oil groups based on distinct physico-chemical characteristics. These lipids were tested on lychee, plums, nectarines, bananas, apple, Nashi and pears. 4.2.2 Results Tropical fruits 4.2.2.1 Lychee Lychee fruit loses its natural red colour rapidly after harvest. The shelf life of unpacked fruit at room temperature is reported to be about 72 hours, due to skin browning (Hau et al., 1979). The possibility of delaying the loss of the red colour as well as reducing weight loss through the use of lipid coatings was examined. 118 Bright red lychees were coated with butter, dripping, peanut, canola, coconut, soybean, sunflower, morakot, castor and liquid paraffin and stored at 20°C. The fruits were assessed for loss of red colour and weight at regular intervals. This study confirmed that lychees lost weight rapidly after harvest. Uncoated lychees stored at 20°C lost about 21% of their initial weight within 7 days (Table 21). In comparison, fruit coated with sunflower, morakot, castor, liquid paraffin, or coconut oil, as well as butter or dripping had only about 13% weight loss after 7 days of storage. Soybean, peanut and canola however, had little effect with an average 19.1% weight loss (Table 21). As red skin colour is the major determinant of the marketable life of lychee, storage life of lychee was defined as time to reach unacceptable browning (score 3, reddish brown). Browning started in uncoated lychees within 2 days of storage at 20°C, and the red colour was completely lost by day 4. Although the red colour of lychee was intensified after coating only lychees coated with castor, dripping or coconut oil retained their red colour longer than the uncoated lychees. Lychees coated with castor oil or dripping on the other hand, retained their red colour for 10 days, while those coated with coconut oil remained red until after 7 days of storage (Table 21). 119 Table 21 Effect of lipid coatings on the weight loss and storage life of lychees at 20°C. Treatment1 Weight loss2 Days to unacceptable browning (%) Uncoated 21.1a 2.5a Saturated lipids Butter 13.2b 3a Dripping 12.4b 10c Monounsaturated lipids Peanut 19.0a 2.5a f Canola 18.4a 2.5a Coconut oil 11.6b 7b Polyunsaturated lipids Soybean 20.1a 2.5a Sunflower 14.5b 4a Marakot 13.6b 4a Castor 12.4b 10b Mineral oil Liquid paraffin 15.1b 3a 1 Three drops of oil and approx. 18 mg of butter and dripping were brushed on the surface of each fruit. 2 Percent weight loss at day 7 of storage. Each value is the mean of 6 replicates of 20 fruits. Values followed by different letters within each column are significantly different at p = 0.05 by DMRT. 120 This experiment shows that dripping, castor and coconut oils were more effective in retaining the red colour of lychee stored at 20°C. Lychee coated with these lipids also had the least weight loss. 4.2.2.2 Plum Plums are of commercial importance with a world production of about 6 million tonnes (Snowdon, 1990). They have, however, a short storage life of about 7 days at ambient temperature and 2 to 4 weeks at 0°C (Salunkhe and Desai, 1984; Snowdon, 1990) depending on the cultivar and stage of fruit maturity. This experiment examined the effect of lipid coatings on the storage life of plum. Santa Rosa plums that were green with slight reddish blotch, were coated with butter, dripping, peanut, canola, coconut, soybean, sunflower, marakot, castor or liquid paraffin and stored at 0° and 20°C. Fruits were assessed for loss in weight, as well as changes in colour, firmness and chemical composition. Plums stored at 20°C were assessed at regular intervals while those kept at 0°C were assessed after 31 days of storage. Table 22 shows the effect of coatings on some quality attributes of plums after 9 days of storage at 20°C. 121 Table 22. Effect of lipid coatings on the physical and chemical characteristics of Santa Rosa plums after 9 days storage at 20°C Treatment1 Weight loss Firmness Colour score TSS Acidity pH (%) (kg) °Brix meq/lOOmL Initial value 2.61 1 11.5 1.27 3.22 Uncoated 2.63 0.63 4.6 14.5 1.05 3.30 Saturated lipids Butter 1.69 0.83 4.8 13.5 0.88 3.4 Dripping 1.71 1.20 4.1 13.25 0.84 3.37 Monounsaturated lipids Coconut 0.93 0.84 4.6 13.25 0.92 3.35 Canola 1.83 0.8 4.7 13.25 0.98 3.32 Peanut 2.11 1.18 4.7 13.5 1.14 3.24 Polyunsaturated lipids Sunflower 1.88 0.74 4.7 13.5 0.94 3.37 Soybean 1.53 0.74 4.8 13.75 0.93 3.29 Castor 1.29 0.91 4.9 11.75 1.0 3.29 Marakot 1.49 1.0 4.7 13.75 1.11 3.25 Mineral oil Liquid paraffin 2.08 0.63 4.8 13 0.97 3.41 LSD (5%) 0.51 0.11 0.22 0.55 0.06 0.12 1 Four drops of oil and approx. 20 mg of butter and dripping were smoothly spread on each fruit Colour score: 1 = green, 2 = < 50% red, 3 = 50 -75% red, 4 = red and 5 = dark red. Each value is the mean of 3 replicates of 20 fruits. 122 Weight loss in uncoated plums was about 2.63% after 9 days of storage at 20°C. Coating significantly (p = 0.05) reduced weight loss with an average loss of 1.6% in coated fruit. Coconut oil was the most effective coating, reducing weight loss to 0.93% (Table 22). Uncoated plums lost about 3.46% of their weight after 31 days of storage at 0°C (Table 23). All coatings significantly reduced (p=0.05) weight loss, with no difference between the 10 lipids studied. Uncoated plums started to turn red after 4 days of storage at 20°C, and were fully red by r day 7. Coating had no effect on delaying colour change, with all coated plums being fully red by day 9 (Table 22). Even though stored at low temperature (0°C), the uncoated fruits were fully red (score 4) after 31 days of storage. Coating delayed colour changes at this temperature, with most coated fruits still at score 3 (50 - 75% red) at the completion of storage (Table 23). The firmness value for plum, as measured by a penetrometer, was 2.61kg at the beginning of storage. Uncoated fruits softened rapidly to 0.63kg within 9 days of storage, whilst retention of firmness was enhanced in all coated fruits (Table 22). Dripping, peanut, marakot, and castor oils were the most effective treatments, reducing softening by 16% compared to the uncoated fruits. 123 Table 23. Effect of lipid coatings on the physical and chemical characteristics of Santa Rosa plums after 31 days storage at 0°C Treatment1 Weight loss Firmness Colour score TSS Acidity pH (%) (kg) °Brix meq/lOOmL Initial value 2.61 1 11.5 1.27 3.22 Uncoated 3.46 0.52 4.4 14.5 0.59 3.74 Saturated lipids Butter 1.25 0.92 2.5 13.5 0.70 3.61 Dripping 1.66 1.16 3.2 12.75 0.91 3.49 Monounsaturated lipids r Coconut 1.69 0.82 3.4 13.25 0.61 3.71 Canola 1.41 1.12 3.2 13.5 0.81 3.55 Peanut 1.65 0.93 3.0 13 0.89 3.48 Polyunsaturated lipids Sunflower 1.52 0.98 3.3 12.75 0.68 3.62 Soybean 1.52 0.95 3.1 12.75 0.68 3.62 Castor 1.69 1.18 3.3 13.5 0.62 3.69 Marakot 1.51 1.39 3.2 12.25 0.83 3.56 Mineral oil Liquid paraffin 1.76 1.05 3.1 13.5 0.60 3.69 LSD (5%) 0.41 0.2 0.5 0.9 0.015 0.02 1 Four drops of oil and approx. 20 mg of butter and dripping were smoothly spread on each fruit Colour score: 1 = green, 2 = < 50% red, 3 = 50 -75% red, 4 = red and 5 = dark red. Each value is the mean of 3 replicates of 20 fruits. 124 Uncoated fruit lost about 80% of their initial firmness after 31 days of storage at 0°C (Table 23). Coated fruits on the other hand remained firmer having lost only 59.7% of their initial firmness. Marakot, castor and canola oils, as well as dripping, were the most effective, being able to reduce softeness by about 27%, as compared to the uncoated plums. The total soluble solids of the uncoated fruit rose from 11.5 to 14.5°Brix after 9 days storage at 20°C, whilst coated fruits were approximately 13.5°Brix (Table 22). Surprisingly acidity fell more in the coated than the non coated plums f A similar result as in 20°C storage, was obtained for TSS changes in plums stored at 0°C but in this case acidity lost was higher in the uncoated than the coated fruits (Table 23). In summary, the coatings used in this experiment did not delay the time for plums to become dark red during storage at 20°C. Softening was, however, delayed by all coatings with the saturated (butter and dripping) and monounsaturated (coconut, canola and peanut) lipids being the most effective. All lipids, however had a similar effect on weight loss, softening and colour loss in plums stored at 0°C. 125 4.2.2.3 Nectarine Shrivelling is recognised as an important marketing problem in nectarines. Shrivelling occurring during handling, storage or distribution, can seriously reduce the sale appeal of the fruit. The purpose of this experiment is to investigate the effect of lipid coatings on storage life of nectarines. Nectarines coated with butter, dripping, peanut, canola, coconut, soybean, sunflower, marakot, castor and liquid paraffin were stored at 0° and 20°C. Fruits were assessed for weight loss, shrivelling and changes in chemical compositions at regular intervals during storage at 20°C and after 29 days of storage at 0°C. Shrivelling was the major form of deterioration in nectarine. Shrivelling started to appear on uncoated nectarines after 4 days of storage at 20°C and became severe by day 6, with fruits becoming dull and flabby. All coated nectarines, on the other hand, were still fresh and showed no shrivelling after 15 days storage at 20°C (Table 24). At 0°C, uncoated nectarines were severely shrivelled (score 4) after 29 days of storage. Although nectarines coated with sunflower, marakot, peanut or coconut oils were slightly shrivelled (score 2), those coated with canola, peanut, castor, liquid paraffin, butter or dripping had no shrivelling (Table 25). 126 Table 24. Effect of lipid coatings on firmness and chemical composition of nectarines after 15 days storage at 20°C Treatment1 Shrivelling Firmness Total Soluble Solid Acidity pH (kg) °Brix meq/lOOmL Initial value 1.34 11 1.15 3.43 Uncoated severe 0.53a 13a 0.75a 3.93a Saturated lipids Dripping nil 0.61b 12b 0.84b 3.82a Butter nil 0.64c 12b 0.85b 3.76a Monounsaturated lipids Coconut nil 0.58b 12b 0.81ab 3.84a Canola nil 0.59b 12b 0.80ab 3.83a Peanut nil 0.63c 12b 0.84b 3.75a Polyunsaturated lipids Soybean nil 0.54a 12.5ab 0.78ab 3.83a Sunflower nil 0.66c 12b 0.86b 3.83a Castor nil 0.68c 12b 0.84b 3.86a Marakot nil 0.69c 11.75b 0.81ab 3.45a Mineral oil Liquid paraffin nil 0.58b 12b 0.83b 3.79a 1 Four drops of oil and approx. 20 mg of butter and dripping were smoothly spread on each fruit Each value is the means of 4 replicates of 10 fruits. Values followed by different letters within each column are significantly different from each other at p = 0.05 by DMRT. 127 Table 25. Effect of lipid coatings on firmness and chemical composition of nectarines after 29 days storage at 0°C Treatment Shrivelling Firmness Total Soluble Solid Acidity pH (kg) °Brix meq/lOOmL Initial value 1.34 11 1.15 3.43 Uncoated severe 0.62a 12a 0.96a 3.73a Saturated lipids Dripping nil 0.69ab 12a 0.94ab 3.73a Butter nil 0.79b 12a 0.85c 3.76a Monounsaturated lipids Coconut slight 0.7b f 12a 0.93ab 3.73a Canola nil 0.79b 12a 0.90ab 3.73a Peanut slight 0.86bc 12a 0.87bc 3.77a Polyunsaturated lipids Soybean nil 0.66a 12a 0.93ab 3.72a Sunflower slight 0.72b 12a 0.96a 3.71a Castor nil 0.87bc 12a 0.95a 3.73a Marakot slight 0.69d 12a 0.87bc 3.77a Mineral oil Liquid paraffin nil 0.72b 12a 0.83c 3.79a 1 Four drops of oil and approx. 20 mg of butter and dripping were smoothly spread on each fruit Each value is the means of 4 replicates of 10 fruits. Values followed by different letters within each column are significantly different at p = 0.05 by DMRT. 128 Weight loss increased rapidly to 8.24% in the uncoated fruit over the first 6 days of storage and was 13.03% by the 15th day (Figure 8). Coating significantly (p = 0.05) reduced weight loss with all coatings having a similar effect. About 50% reduction in weight loss was achieved in coated fruits throughout the entire storage period at 20°C (Figure 8). At 0°C, weight loss in uncoated nectarines was 9.08% after 29 days of storage (Figure 9). This was also significantly (p = 0.05) reduced by all coatings. Liquid paraffin, canola and castor oils were the most effective treatments (Figure 9), reducing weight loss by r about 64.5%. Firmness in the uncoated nectarines decreased from 1.34kg to 0.53kg after 15 days storage at 20°C. Although all coated nectarines were firmer than the uncoated, only those coated with butter, peanut, sunflower, castor and marakot oils were statistically different (p=0.05) from the uncoated fruit (Table 24). After 29 days of storage at 0°C, firmness decreased in uncoated nectarines to 0.62kg. All coatings except for dripping and soybean significandy (p = 0.05) delayed softening of the nectarines (Table 25). 129 Storage time (days) Figure 8. Effect of lipid coatings on weight loss of nectarines stored at 20°C. ( ■-) Uncoated and (°") mean values of nectarines coated with dripping, butter, canola, peanut,, marakot, soyabean, castor, liquid paraffin, sunflower and coconut oils. Figure 9. Effect of lipid coatings on weight loss of nectarines after 29 days storage at 0°C Ctl = Control; ST = saturated lipids(dripping and butter); MU = monounsaturated (canola, peanut and coconut oils); PU = polyunsaturated (marakot, soybean, castor and sunflower) and MO = mineral oil (liquid paraffin) 130 Total soluble solids increased from 11° to 13°Brix in uncoated nectarines after 15 days of storage at 20°C. Increase in TSS was delayed in all coated fruits with most of them remaining at 12°Brix after 15 days (Table 24). Acidity was drastically decreased in the uncoated fruit from 1.15 to 0.75 meq/lOOmL after 15 days. This decrease was significantly (p = 0.05) delayed in all coated fruits, especially in those coated with canola, marakot, soybean and coconut oils (Table 24). Nectarines coated with these oils had an acidity value of 0.8 meq/lOOmL after 15 days of storage. The initial pH value at storage was 3.43, and was 3.93 in the uncoated fruit after 15 days. There was no marked (p = 0.05) effect of coating on delaying changes in pH. Coating had no great effect on the chemical composition of coated nectarines after 29 days of storage at 0°C (Table 25). In summary, shrivelling which is related to loss of water from the fruit, was noticeably delayed by all coatings in nectarines stored at both 0° and 20°C. All lipids used in this experiment reduced weight loss by half during storage at 20°C, subsequently preventing shrivelling of the fruit. Weight loss was also reduced at 0°C, with some coatings still allowing slight shrivelling on the fruit. This could be due to the relative humidity of the storage room which was about 80%. The use of lipids as coatings for nectarines appears promising and could be of importance in solving the marketing problem associated with stored nectarines. 131 4.2 2.4 Banana Bananas are commercially important fruit crop in world trade (Salunkhe and Desai, 1984). Perishable commodities like bananas spoil quickly unless given special treatments such as cold and modified atmosphere storage. Although bananas are consumed ripe, they must be shipped unripe for export. The possibility of using lipid coatings to extend the storage life of banana is considered in this experiment. Green bananas coated with butter, dripping, peanut, canola, coconut, soybean, sunflower, marakot, castor and liquid paraffin were stored at 7° and 20°C. Fruits were assessed for weight loss and colour changes at regular intervals during storage at 20°C and for symptoms of chilling injury after storage at 7°C. Uncoated bananas started to degreen after 4 days storage at 20°C and became fully yellow after 11 days of storage (Figure 10). Coated bananas, on the other hand, retained the full green colour longer. Liquid paraffin, castor oil, dripping and butter were more effective, retaining the full green colour until day 7, compared to about 5 days in those coated with peanut, soybean, canola, sunflower, marakot or coconut oil (Figure 10). Full ripening was significantly delayed (p=0.05) in bananas coated with peanut, soybean, canola, sunflower, marakot or coconut oils with fruit attaining a full yellow colour and softened flesh after 15 days, compared to 10 days in the uncoated fruit (Figure 10). Bananas coated with liquid paraffin, castor, dripping and butter however, did not attain full yellow colour by the end of the storage period although the fruit flesh was softened. 132 Storage time (days) Figure 10. Effect of lipid coatings on colour changes of bananas stored at 20°C. (-H-) Control; (40 average values for bananas coated with peanut, soybean, canola, sunflower, marakot, and coconut oils that attained full yellow colour by the end of 19 days storage; and (-0) average values for bananas coated with liquid paraffin, castor, dripping and butter that did not attain full yellow colour at the end of storage. Colour scores: 1 = green to 8 = yellow with increased brown areas. Minimum acceptability = score 6 (full yellow) 133 Plate 2 shows the effect of the lipid coatings on bananas after 18 days of storage at 20°C. Uncoated bananas lost about 8.4% of its weight within 12 days of storage at 20°C (Figure 11). Weight loss was reduced significantly (p=0.05) by the lipid coatings, with no difference between treatments. An average weight loss of 7.01% was obtained in the coated banana after 12 days of storage. The appearance of "flecking", a postharvest disease associated with ripening bananas and caused by Colletotrichum sp., occurred on uncoated banana after 10 days of storage and was severe by day 12. This was significantly (p = 0.05) delayed by coating, with flecking remaining slight on bananas coated with peanut, soybean, canola, sunflower, marakot and coconut oils after 17 days storage. Flecking was prevented on bananas coated with liquid paraffin, dripping, castor and butter. Bananas stored at 7°C were assessed for chilling injury after 5 days storage. Chilling injury was severe on uncoated bananas, appearing as smoky discolouration of the peel. The smoky discolouration of the peel was absent on bananas coated with marakot, soybean, canola, coconut, butter and dripping (Plate 3) after removal from the cold storage. Fruits however did not ripen uniformly when subsequently held at 20°C. In this experiment there was a delay in the ripening, as assessed by colour and softeness, of bananas coated with peanut, soybean, canola, sunflower, marakot or coconut oil. 134 Plate 2. Coated bananas after 18 days storage at 20°C. LQ: liquid paraffin; BT: butter; CS: castor oil; CO: coconut oil; DR: dripping; PN: peanut oil; CA: canola; SN: sunflower oil; SB: soybean; Thai: marakot oil 135 20 15 - - 10 - CD CD £ 5 - 5 10 Storage time (days) Figure 11. Effect of lipid coatings on weight loss of bananas stored at 20°C. (-■-) Uncoated and (mean values for bananas coated with dripping, butter, canola, peanut, marakot, sunflower, soyabean, castor, liquid paraffin and coconut oils. 136 Plate 3. Effect of lipid coatings on chilling injury of bananas after 1) 3 days storage at 7°C and 2) + 4 days at 20°C. 137 Bananas coated with liquid paraffin, castor, dripping and butter did not ripen properly, making the lipids unsuitable for the storage of bananas at 20°C. Since flavour is a very important parameter in the quality of banana (Banks, 1984; Olorunda and Aworh, 1984), further work involving sensory analysis will be needed to establish the promising effect of lipid coatings on banana storage at 20°C. Pome fruits 4.2.2.5 Apple Apple, a temperate climacteric fruit, is the most commonly consumed fruit in Australia (HRDC, 1990; Yuen et al., 1994). Apples are generally stored at temperatures around 0°C (Snowden, 1990). Long-term storage at these temperatures can result in physiological disorders such as superficial scald, core and flesh browning, core flush and low temperature injuries, depending on the variety. Earlier works on coating Granny Smith apples with oils delayed senescence but resulted in injury and off-flavour when 10% castor oil and shellac was used (Trout et al, 1957). Smaller amounts (6 drops) was used in this experiment with the aim of extending the storage life of Granny Smith apples at both 0° and 20°C. 138 Green Granny Smith apples coated with butter, dripping, peanut, canola, coconut, soybean, sunflower, marakot, castor oils or liquid paraffin were stored at 0° and 20°C. Fruits were assessed for weight loss and changes in colour and chemical composition at regular intervals during storage at 20°C. Apples were assessed for physiological disorders after 17 weeks of storage at 0°C and 1 week at 20°C. The desired colour of Granny Smith apple is green, so acceptability was based on green colour retention. Uncoated apples retained full green colour for 3 weeks during storage at 20°C. Degreening started after 3 weeks and uncoated apples became unacceptable (colour score 3, 50% yellow) after 8 weeks. In comparison, all coated apples retained the full green colour (score 1) for 17 weeks. Plate 4 shows the effect of coatings on green colour retention of Granny Smith apple after 7 and 13 weeks storage at 20°C. Firmness of fruit decreased significantly (p=0.05) during storage, changing from an initial value of 5.84kg to 4.59kg in the uncoated apple after 53 days of storage at 20°C (Figure 12a). Coating significandy reduced (p=0.05) softening with the fruits having a firmness value of 5.13kg after 53 days (Figure 12a) with very little difference (p=0.05) between the different coatings (Table 26). 139 Plate 4. Coated Granny Smith apples after a) 7 weeks and b) 13 weeks storage at 20°C. PN: peanut oil; SB: soyabean oil; SN: sunflower oil; CA: canola oil; Thai: marakot. 140 6 Storage time (days) Figure 12. Effect of lipid coatings on (a) firmness and (b) weight loss of Granny Smith apple stored at 20°C. (* ) Uncoated and (a-) mean values of apples coated with dripping, butler, coconut, peanut, canola, castor, soybean, sunflower, marakot, and liquid paraffin oil Vertical bars represent standard errors of the means. 141 Table 26. Effects of lipid coatings on firmness and chemical composition of Granny Smith apples after 53 days storage at 20°C Treatment ^ Firmness Acidity PH Total Soluble Solid (kg) meq/lOOmL (°Brix) Initial value 5.84c 0.56a 3.33c lib Uncoated 4.59a 0.39b 3.48ab 12.75a Saturated lipids Dripping 5.04ab 0.43b 3.48ab 13.25a Butter 4.97ab 0.36a 3.52ab 12.5a Monounsaturated lipids Coconut 5.23b 0.42b 3.48ab 13a Peanut 5.24b 0.39b 3.49ab 13a Canola 5.27b 0.42b 3.46b 12.75a Polyunsaturated lipids Castor 5.01ab 0.37b 3.5ab 12.75a Soybean 5.01ab 0.40b 3.46b 12.75a Sunflower 5.1 lab 0.41b 3.46b 13a Marakot 5.26b 0.37b 3.47ab 13a Mineral oil Liquid paraffin 5.19b 0.39b 3.49ab 12.75a 1 Six drops of oil and approx. 25mg of butter and dripiing were smootnly spread on each fruit. Each value is the mean of 6 replicates of 20 fruits. Values followed by different letters within a column are significantly different from each other at p = 0.05 by DMRT. 142 Weight loss, though not a serious problem during prolonged storage of Granny Smith apples, was about 5.97% in the uncoated fruit after 61 days of storage at 20°C. This was reduced by an average of 18.6% in all coated apples (Figure 12b). TSS and pH of uncoated apples significantly (p = 0.05) increased from an initial ll°Brix and 3.33 to 12.75°Brix and 3.48, respectively, after 53 days of storage (Table 26), while the acidity decreased from an initial 0.56 meq/lOOmL to 0.39 meq/lOOmL. Although the coatings had no marked effect (p = 0.05) on the chemical composition of apples (Table 26), all coated fruits were considered crispier and juicer based on personal subjective evaluation. Table 27 presents the values for per cent shrivelling and rots observed on coated fruits after 81 days storage at 20°C. There was no shrivelling observed on uncoated apples at the completion of storage. Coating the fruit however, encouraged shrivelling. Apples coated with peanut, soybean or butter were severely shrivelled while those coated with liquid paraffin, marakot, canola, sunflower or castor oil had substantially lower levels of shrivelling. About 20% of the uncoated apples developed rots after 81 days. Incidence of rot was higher in fruits coated with soybean, liquid paraffin or coconut oil but reduced in those coated with sunflower, castor or dripping coatings (Table 27 ). 143 Table 27. Shrivelling and rotting of Granny Smith apples after 81 days storage at 20°C Treatment Shrivelling Rot (%) (%) Uncoated 0 20 Saturated lipids Dripping 31.6 10 Butter 72 20 Monounsaturated lipids Peanut 50 30 Coconut 33.3 40 Canola 20 20 Polyunsaturated lipids Sunflower 18.75 10 Castor 20 10 Marakot 18.75 30 Soybean 53 40 Mineral oil Liquid paraffin 10 40 LSD (5%) 12.1 10.2 Each value is the mean of 6 replicates of 20 fruits 144 Apples were assessed for physiological disorders after 17 weeks storage at 0°C and an additional 9 days at 20°C. Both coated and uncoated apples remained green with no shrivelling throughout storage at 0°C. Superficial scald developed only on the uncoated fruit, with about 95.5% of the fruit being affected. The uncoated fruit were severely scalded (score 5) while all coated fruits had no superficial scald (score 1). Plate 5 shows the effect of the coatings on the development of superficial scald on Granny Smith apples stored at 0°C. 145 Plate 5. Granny Smith apples coated with different oils after 17 weeks storage at 0°C + 1 week at 20°C. PN: peanut oil; SB: soybean oil; SN: sunflower )il; CA: canola oil; Thai: marakot. 146 In summary yellowing was delayed by all coatings with coated apples retaining the green colour 9 weeks longer than in the uncoated fruits during storage at 20°C. Shrivelling was however, a problem in the coated fruits and was severe after 81 days of storage at 20°C in some coated fruits. Sunflower and castor oil had the lowest levels of shrivelling and rot. Castor oil, however, gave an unnatural glossiness to the apples, so sunflower oil may have some potential with apples at 20°C. Superficial scald was prevented by all lipid coatings. 4.2.6 Nashi (Japanese pear) Nashi is an exotic fruit whose crispy and refreshing nature has made it a delicacy to be enjoyed chilled and sliced. This fruit is native to Asia and is fast becoming popular in Australia. Shrivelling, yellowing and water loss are the major causes of quality loss during lengthy periods of cold storage (Johnson, 1989). Physiological disorders can also develop. This experiment was designed to examine the effect of lipid coatings on the storage life of Nashi at 0° and 20°C. Green Nashi fruits treated with butter, dripping, peanut, canola, coconut, soybean, sunflower, marakot, castor oils or liquid paraffin were stored at 0° and 20°C. Fruits 147 were assessed for colour, weight loss and changes in chemical composition during storage. Nashi stored at 0°C were also assessed for physiological disorders after 24 weeks of storage and an additional 1 week at 20°C. Nashi fruit change from green to yellow during storage. Uncoated fruits started to yellow after 5 days storage at 20°C and were fully yellow (score 5) after 17 days (Figure 13a). All coatings were highly (p=0.05) effective in delaying the yellowing of Nashi stored at 20°C. The mean value for all the coatings showed coated fruits to be only 25% yellow (score 2) after 17 days of storage, (Figure 13a). Plate 6 and 7 show the effect of coating on the colour change of Nashi stored at 0° and 20°C. 148 0 5 10 15 20 25 30 35 Storage time (days) Figure 13. Effect of lipid coatings on (a) colour, (b) firmness and (c) weight loss of Nashi stored at 20°C. (-») Uncoated and (-o-) mean values of Nashi coated with dripping, butter, coconut, peanut, canola, castor, soybean, sunflower, marakot, and liquid paraffin oil. Colour score: 1 = green, 3 = 50% yellow and 5 = full yellow. Vertical bars represent standard errors of the means. 149 ♦ Control Sovabean >'-dor Coconut Butter Canola jr* *- P^| u, , j Control I iquid I’aratlm ' T I hailandCTT I’eanut | Sunflower^^J_[^PP^ Nashi treated with different oils after 19 days storage at 20'C. Plate 6. Nashi coated with different lipids after 19 days storage at 20°C. 150 Pilate 7. Nashi coated with different oils after 28 weeks storage at 0°C + 1 week at 20°C. SN: sunflower oil; LQ: liquid paraffin; DR: dripping; PN: peanut oil; CA: canola oil; CS: castor oil; BT: butter; SB: soybean oil; Thai: marakot; CO: coconut oil. 151 Although stored at 0°C, uncoated Nashi were fully yellow after 24 weeks storage at 0°C. Coating significantly delayed colour change with coated fruits retaining at least 50% of their initial green colour, even after 7 days storage at 20°C (Table 28). Firmness of the untreated fruit decreased from an initial value of 4.54 to 2.31kg after 15 days of storage at 20°C. Coating significantly (p=0.05) delaying softening with coated fruit remaining firmer at 4.15kg (Figure 13b). At 0°C, uncoated Nashi lost about 60% of its initial firmness after 24 weeks storage with a firmness value of 1.8kg compared to the initial 4.54kg. Coated fruits however, remained firmer delaying softening by 34.6% (Table 28). Weight loss increased with storage time but was significantly (p=0.05) reduced by the coatings (Figure 13c). Weight loss in the uncoated fruit was 5.83% after 27 days of storage at 20°C. Reduction in weight loss was achieved by the coatings, with an average reduction of 22.8%. Weight loss in uncoated Nashi was 12.8% after 24 weeks of storage at 0°C. This was significantly reduced to between 3.9 - 7.6% in all coated fruits. Total soluble solids (TSS) and pH of uncoated Nashi increased from an initial ll°Brix and 4.36 to 12°Brix and 4.75, respectively, after 15 days of storage at 20°C (Table 29). Coating, had little effect (p=0.05) on delaying changes in TSS and acidity of Nashi (Table 29). 152 Table 28. Effect of lipid coatings on physical and physiological changes of Nashi after 24 weeks storage at 0°C. Treatment1 Weight loss Colour score Core browning Firmness (%) score (%) (kg) Initial - 1 - 4.54 Control 12.8 4.9 3.03 (83.3) 1.83 Saturated lipids Dripping 4.8 2.4 1.70 (30.8) 4.01 Butter 5.5 2.5 1.25(19.5) 3.29 Monounsaturated lipids Peanut 4.0 2.5 1.34(17.6) 3.51 Coconut 4.6 2.5 1.28(12.5) 3.09 Canola 5.4 2.3 1.37 (25) 3.28 Polyunsaturated lipids Sunflower 4.3 2.6 1.39 (21.6) 3.27 Soybean 5.4 2.3 1.38 (28.9) 3.24 Castor 5.8 1.9 1.74 (28.9) 3.28 Marakot 7.6 2.5 1.49 (30) 3.67 Mineral oil Liquid paraffin 3.9 2.3 1.39 (20.5) 3.36 LSD (5%) 0.22 0.31 0.41 0.69 1. Six drops of oil and approx. 25 mg of butter and dripping were spread over each fruit. Colour score: 1 = green, 2 = < 25% yellow, 3 = 25 - 50% yellow, 4 = 50 - 75% yellow and 5 = fully yellow. Core browning: 1 = none, 2 = slight browning, 3 = moderate browning, 4 = severe browning and 5 = black. Values in parentheses are % affected fruits. Each value is the mean of 6 replicates of 20 fruits. 153 Table 29. Effect of lipid coatings on changes in chemical compositions of Nashi after 15 days of storage at 20°C Treatment1 Total Soluble Solids Acidity pH °Brix meq /100mL Initial 11 0.34 4.18 Control 12a 0.26a 4.39a Saturated lipids Butter 11.5ab 0.23b 4.5b Dripping lib 0.23b 4.53b Monounsaturated lipids Coconut lib 0.21b 4.64d Peanut 11.5ab 0.22b 4.61cd Canola lib 0.21b 4.58c Polyunsaturated lipids Castor 11.25ab 0.21b 4.63d Marakot 11.25ab 0.21b 4.68e Soybean 11.25ab 0.17c 4.73f Sunflower 11.25ab 0.21b 4.65d Mineral oil Liquid paraffin 11.25ab 0.21b 4.65d 1. Six drops of oil and approx. 25 mg of butter and dripping were spread over each fruit. Each value is the mean of 6 replicates of 20 fruits. Values followed by different letters within a column are significantly different from each other at p = 0.05 by DMRT. 154 Coated fruit maintained the desirable taste and flavour and were more juicy than the control, based on personal subjective evaluation. Nashi were removed from 0°C after 24 weeks of storage and held for 7 days at 20°C for physiological disorders to develop. The only physiological disorder observed in uncoated Nashi after cold storage was core browning. About 83.3% of the uncoated fruit were affected, with a mean severity score of 3.03 (moderate browning). The cores of affected fruit were brown and in some f severe cases black. Coating maikedly (p = 0.05) reduced the severity of core browning to an average score of 1.43 (slight browning) in the coated fruit with only about 3.2% of fruit affected (Table 28). In summary, storage life, assessed by overall marketability (colour, taste and firmness), was 17 days at 20°C in uncoated Nashi. This was extended to 33 days in all coated fruit and the coatings gave the fruit an appealing glossiness. In respect to overall acceptability, all the lipids used in this experiment are promising in the storage of Nashi at both 0° and 20°C, except for dripping and butter which were sticky, and castor oil which was too glossy and caused lenticel injury. 155 4.2.7 PEARS (EUROPEAN) Pears like most other temperate fruits are better stored at temperatures around 0°C. Pears are being stored in conventional air storage at -1°C, as well as controlled atmosphere (CA) (Chen et al (1990). Under both storage conditions, superficial scald was reported as a major physiological disorder which affects the external appearance of some pear varieties during the marketing period. This work was undertaken to assess the potential of lipid coatings for preserving quality and extending the storage life of Packham pears. Green Packham Triumph pears were coated with butter, dripping, peanut, canola, coconut, soybean, sunflower, marakot, castor oils or liquid paraffin and stored at 0° and 20°C. Fruits were assessed for colour, weight loss and changes in chemical composition during storage. Pears stored at 0°C were also assessed for physiological disorders after 30 weeks of storage and 9 days at 20°C. The colour index in the uncoated pears increased from 1 (green) at harvest to 4 (yellow) after 15 days storage at 20°C, with fruit becoming very soft after 21 days of storage. Coating significandy (p = 0.05) delayed yellowing of pear, with all coated fruit still remaining green (score 1-2) after 34 days (Figure 14). Browning of the skin appeared at this stage and rendered all coated pears unacceptable after 40 days of storage. 156 Figure 14. Effect of lipid coatings on colour change of pears stored at 20°C. (-*-) Uncoated and (-&) mean values of pears coated with dripping, butter, coconut, peanut, canola, castor, soybean, sunflower, marakot, and liquid paraffin oil. Vertical bars represent standard errors of the means. 157 Plate 8 shows the effect of some oil coatings on pears during storage at 20°C. Storage at 0°C delayed colour changes in pears (Table 30). Uncoated fruits were just starting to change from green to yellow (score 3) after 30 weeks storage and were fully yellow after a further 9 days storage at 20°C. Colour change was delayed by all coatings with a mean colour score of 2 (25% yellow) after 30 weeks storage at 0°C, with greenness still maintained after 9 days at 20°C. Uncoated pears lost about 3.8% of their weight after 17 days storage at 20°C (Figure f 15). This was significantly reduced (p=0.05) to 2.1% in coated fruits, achieving a 46% reduction. Firmness of the pears decreased during storage, changing from an initial 7.66kg to 1.96kg in uncoated fruit after 15 days storage (Table 31). Coating significantly (p=0.05) reduced softening with peanut, soybean, sunflower, canola, marakot and castor oils being more effective. Pears coated with these oils had an average value of 7.04kg. Although yellowing of the uncoated fruit was delayed by the low temperature storage, the flesh of the fruit softened considerably, with firmness significandy reduced from an initial 7.66kg to 1.20kg (Table 30) after 30 weeks storage at 0°C. Coated fruits, on the other hand remained firmer with an average value of 5.81kg. 158 COntrol COc omit Castor Liquid Paraffin Butter 1 Dripping Plate 8. Pears coated with different lipids after 22 days of storage at 20°C. 159 Table 30. Effect of lipid coatings on physical and physiological changes of Packham Triuimph pears after 30 weeks storage at 0°C. Treatment Firmness TSS Acidity pH Colour score Superficial (kg) °Brix scald score Initial 8.2 11 0.107 4.21 1 1 Control 1.20 13.37 0.145 4.46 3.15 4.4 Saturated lipid Butter 5.81 12.62 0.14 4.47 2.1 2.08 Dripping 5.76 12 0.13 4.49 2.0 2.08 Monounsaturated lipid Coconut 4.94 12 0.13 4.5 1.83 1.67 Peanut 5.53 12.62 0.1,45 4.4 2.15 1.8 Canola 6.42 12.19 0.16 4.32 2.02 2.0 Polyunsaturated lipid Castor 5.54 12 0.13 4.57 2.3 1.5 Marakot 5.97 12 0.17 4.4 2.25 2.0 Soybean 5.85 12.5 0.15 4.46 2.05 1.95 Sunflower 5.84 12.62 0.12 4.54 2.42 1.9 Mineral oil Liquid paraffin 6.82 12 0.13 4.58 2.1 1.85 LSD (5%) 0.37 0.73 0.045 0.02 0.106 0.61 Six drops of oil and approx. 25 mg of butter and dripping were spread over each fruit. Colour score: 1 = green, 2 = < 25% yellow, 3 = 25 - 50% yellow, 4 = 50 - 75% yellow and 5 = fully yellow. Superficial scald; 1 = none, 2 = slight browning, 3 = moderate browning, 4 = severe and 5 = very severe. Each value is the mean of 6 replicates of 20 fruits. 160 iO V) o jc CD CD Storage time (days) Figure 15. Effect of lipid coatings on weight loss of pears stored at 20°C. ( »i Uncoated and (-0 ) mean values of pears coated with dripping, butter, coconut, peanut, canola, castor, soybean, sunflower, marakot, and liquid paraffin oil. Vertical bars represent standard errors of the means. 161 Table 31. Effects of lipid coatings on firmness and chemical composition of Packham Triumph pears after 15 days storage at 20°C Treatment Firmness Total Soluble Solids Acidity pH (kg) (°Brix) meq/lOOmL Initial 8.2 11 0.107 4.21 Control 1.54 13.75 0.14 4.56 Saturated lipids Butter 5.88 12.75 0.19 4.35 . Dripping 6.03 12.75 0.17 4.53 Monounsaturated lipids Coconut 5.59 12.87 0.19 4.4 Peanut 6.85 12.75' 0.22 4.29 Canola 7.37 12.5 0.17 4.54 Polyunsaturated lipids Castor 6.83 12.5 0.19 4.45 Marakot 6.88 12.5 0.22 4.51 Soybean 6.97 13.0 0.20 4.30 Sunflower 7.33 12.75 0.18 4.52 Mineral oil Liquid paraffin 4.42 12.75 0.18 4.34 LSD (5%) 0.93 0.255 0.05 0.051 Six drops of oil and approx. 25 mg of butter and dripping were spread over each fmit. Each value is the mean of 6 replicates of 20 fruits. 162 Changes in total soluble solids, acidity and pH were significantly delayed by all coatings during storage at 20°C (Table 31). There was a great variation within the effect of the different lipids on acidity and pH during storage at 0°C although they all had a similar effect on the total soluble solids. The coatings however, had little effect on the chemical composition relative to the uncoated fruit (Table 30). The pears were assessed for physiological disorders after 30 weeks storage at 0°C and 9 days at 20°C. Superficial scald developed on all stored fruit but was reduced (p=0.05) by the coatings (Table 30). Uncoated pears were severely scalded (score 4) with 80% of « the fruits affected. Scald was significantly reduced (p=0.05) with coated fruits being moderately scalded with about 22 - 34% affected fruits. Plate 9 illustrates the effect of lipid coatings on superficial scald of Packham Triumph pears. 163 Plate 9. Pears coated with different oils after 30 weeks storage at 0°C + 12 days at 20°C. SN: sunflower oil; LQ: liquid paraffin; DR: dripping; PN: peanut oil; CA: canola oil; CS: castor oil; BT: butter; SB: soybean oil; Thai: marakot; CO: coconut oil. 164 The delay in changes in colour, firmness and chemical composition indicated that the lipid coatings were able to delay ripening of pears stored at both 20 and 0°C. The coatings, however, did not allow for full yellowing of the pears before browning of the skin started. This may reduce the value of these lipid coatings on pears meant for fresh consumption. Further studies could be done to determine the amount of coating to be used. Dripping and butter coatings were sticky throughout storage therefore could not be recommended. Liquid paraffin, coconut, castor and peanut oils gave a better result than the other coatings in reducing scald at 0°C. 165 4.3 Formulation trials Emulsions were formulated using different combinations and concentrations of food ingredients as previously shown in Table 14 (Section 3.3). Sunflower oil emulsion containing oil-in water at ratios 1:1, 1:3 and 1:5 were trialed on avocado, bittermelon, carrot, cucumber, green beans, kiwi fruit, parsnips, pepper (chilli), snow peas, tamarillo and zucchini. Table 32 summarises the quality of the coated produce after the control produce were no longer acceptable. The number of days in parentheses indicate the storage life of the control produce. t Table 32. Formulation trials on selected produce Produce 1:1 oil/water emulsion 1:3 oil/water emulsion 1:5 oil/water emulsion Beans completely shrivelled 40% of coated beans 40% of coated beans (3 days) and mouldy were shrivelled, were shrivelled, 60% acceptable 60% acceptable Bittermelon glossy, emulsion in emulsion within 33.3% of coated (6 days) crevices, 66.7% of crevices, 33.3% of bittermelons had coated bittermelon coated bittermelons split open, 66.7% were shrivelled had started to split were still acceptable open while 66.7% were mouldy Yellow squash 66.7% of coated 66.7% of coated all coated squash (4 days) squash had light squash were mouldy remained acceptable brown patches and were mouldy 166 Table 32 (contd.) Produce 1:1 oil/water emulsion 1:3 oil/water emulsion 1:5 oil/water emulsion Pepper - chilli coated pepper were coated pepper coated pepper had a (5 days) too glossy with gum remained green with uniform red/orange and oil patches, all 66.7% of vegetable colour with moderate vegetable were shrivelled shrivelling severely shrivelled Zucchini 66.7% of coated 33.3% of coated all coated zucchini (4 days) zucchini were rotten zucchiniwere rotten remained acceptable Tamarillo All coated fruits were rotten after 5 days of storage r (7 days) Cucumber all coated cucumbers all coated cucumbers all coated cucumbers (6 days) remained acceptable remained acceptable remained acceptable Avocado fruits remained hard, fruits remained hard uneven ripening and (3 days) with internal rot with internal rot mouldiness Kiwi all coated fruits all coated fruits all coated fruits (5 days) remained acceptable remained acceptable remained acceptable Snowpea water-soaked water-soaked all coated snowpea (4 days) appearance with appearance and remained acceptable brown discolouration mouldy from coating, slimy and mouldy Carrot brown discolouration brown discolouration moderately shrivelled (9 days) Parsnips all coated parsnips all coated parsnips 33.3% were slightly (9 days) were wilted, sticky, were slightly sticky wilted and had started with brown with 66.7% sprouted sprouting discolouration 167 In summary, it was observed that emulsions containing 20 - 50% oil-in-water had an adverse effect on most of the produce tested. Emulsions of 1:5 (<20% oil) oil-in-water, however, gave promising results with bittermelon, yellow squash, pepper, zucchini, cucumber, kiwi fruit, snowpea and carrot. Lower concentrations of oils were indicated from these results so stock emulsion containing not more than 10% sunflower oil was then prepared as outlined in Section 3.3 and trialed at different concentrations on fewer number of produce 168 4.4 Effect of oils and oil emulsions in extending the storage life of produce 4.4.1 Introduction Properties such as the degree of saturation of the lipids used in Section 4.2 appeared to have no influence on their ability to extend the storage life of fruits and vegetables. Sunflower and marakot oils were chosen for subsequent experiments as they are the most readily available commercial oils in Australia and Thailand. Coconut oil was also chosen because of its non greasy appearance on produce after coating. The use of oil/water emulsions was investigated as a means of lowering the concentration of oil as well as improving the method of application. The stock emulsion was formulated as previously discussed in Section 3.3 using sunflower oil, gum tracaganth, Agral and water, and subsequently diluted to the required concentrations. These experiments were designed to examine the effect of oils and oil coatings on the storage life of cucumber, avocado, apple and Nashi. 4.4.2 Result 4.4.2.1 Cucumber Coconut, marakot, sunflower oils and two emulsions containing 2.5 and 5% (w/w) sunflower oil were tested on green cucumbers at 20°C. Six drops of oil were smoothly spread on each cucumber while the emulsions were appliedd as dip. Untreated cucumbers started to yellow after 13 days of storage at 20°C and were unacceptable by day 16. Only the ‘pure’ oil coatings delayed the loss of green colour in cucumbers. Cucumbers coated with marakot or sunflower oil developed an undesirable yellow colour (score 3, 25% yellow) after about 29 days of storage, compared to 16 days in untreated cucumbers (Figure 16). 169 6 ~ 20 Storage tim6 (days Figure 16. Effect of oils and oil emulsions on colour, shrivelling and weight loss of cucumber stored at 20°C (*-) Uncoated; (o) Coconut oil; (^) Marakot; (-0) Sunflower; (-dr) 5% (w/w) sunflower emulsion and (-&0 2.5 % (w/w) sunflower emulsions. Colour score : 1 = dark green, 3 = 50% yellow and 5 = full yellow. Shrivel score : 4 = no shrivelling, 3 = slight, 2 = moderate and 1 = severe. 170 The oil emulsions had an adverse effect on colour, accelerating the loss of green colour (Figure 16), with cucumbers dipped in emulsions containing 2.5 or 5% (w/w) sunflower oil emulsions becoming unacceptable after 14 days of storage, 2 days earlier than the uncoated produce. Plate 10 shows the effect of oil coating on the colour of cucumber after 28 days storage at 20°C. Shrivelling became visible on the uncoated cucumbers after 6 days of storage at 20°C and was at the minimum acceptable level (moderate shrivel, score 2) after 16 days (Figure 16). Cucumber coated with Marakot or sunflower oil remained just slightly t shrivelled (score 3) after 26 and 32 days, respectively. The emulsions in contrast, accelerated shrivelling, with cucumbers dipped in emulsion containing 2.5 and 5% (w/w) sunflower oil becoming unacceptable at day 16. Weight loss in untreated cucumbers was about 17% after 39 days storage at 20°C. Coconut oil, although not able to delay shrivelling and reduced the loss of green colour, was the only coating effective in reducing weight loss. All treated cucumbers, except those coated with coconut oil, had a higher weight loss than the untreated produce (Figure 16). Weight loss in cucumbers treated with coconut oil was about 13% compared to 17% for the uncoated cucumbers after 39 days storage at 20°C. There was an average weight loss of about 18% in cucumbers dipped in 2.5% (w/w) sunflower oil emulsion, or coated with marakot or sunflower oil and a weight loss of 25.7% in those dipped in 5% (w/w) sunflower emulsion. Shrivelling and colour change were the predominant attributes determining the quality of the cucumbers. 171 Plate 10. Coated cucumbers after 28 days of storage at 20°C. CO: coconut oil; SN: sunflower oil; MKT: marakot oil. 172 Storage life, assessed as time to first reach colour score 2 (25% yellowing) or shrivelling score 2 (moderate shrivelling), was 16 days in the untreated cucumbers (Table 33). This was extended by 10 and 16 days in cucumbers coated with marakot or sunflower oil, respectively. Storage life was however reduced by 2 days in cucumbers dipped in 2.5 and 5% (w/w) sunflower oil emulsions (Table 33). Sunflower and marakot are, therefore, the only coatings (studied) that can be considered as having any potential for extending the storage life of cucumber stored at 20°C. Table 33 Effect of oil and oil emulsions on the storage life of cucumbers stored at 20°C Treatment1 Storage life2 Untreated 16b Sunflower oil 30d Marakot oil 28c Coconut oil 16b 5% (w/w) sunflower emulsion 14a 2.5% (w/w) sunflower emulsion 14a 1 Oils were applied 6 drops/fruit while emulsions were applied as a 10 s dip. 2 Storage life was determined as time to first reach colour score 2 (25% yellow) or moderate shrivelling (score 2). Values followed by different letters are significantly different from each other at p=0.05 by DMRT. Each value is the mean of 6 replicates of 20 fruits. 173 4.4.2.2 Avocado Emulsions containing 0.6, 1.6, 2.5 and 5% (w/w) sunflower oil were evaluated on avocadoes at 20°C. These emulsions were applied by dipping. Weight loss of untreated fruit at 20°C increased with storage time (Figure 17) and reached 9.3% after 18 days storage. All emulsions significantly (p = 0.05) reduced weight loss with emulsions containing 2.5 and 5% (w/w) sunflower oil being more effective, with an average weight loss of 6.03% after 18 days storage at 20°C (Figure 17). The different emulsion concentrations had no effect on the change of skin colour from green to purple during storage. The skin colour of treated and untreated avocados changed from green to purple/brown within 11 days (Figure 17). Although treated fruits attained full ripeness colour (score 1), softening was delayed by the application of coatings. Uncoated avocados were very soft with a firmness score of 1 after 18 days storage. All coated fruit, especially those coated with high concentrations of the emulsions (2.5 and 5% w/w oil), remained firmer and had a firmness score of 3 (Figure 17) while those coated with 0.6 and 1.6% w/w sunflower emulsions were moderately soft (score 2) at the completion of storage. Table 34 shows the effect of coatings on the time to optimum eating quality of avocado, determined as moderate softeness. This was about 10 days in uncoated fruit and was extended by all coatings. Fruits dipped in 5% (w/w) sunflower oil emulsion were still firm when the experiment was terminated. It was considered that normal ripening would probably not occur with further storage at 20°C. 174 10 4 -ti-* Storage time (days) Figure 17. Effect of oil emulsions on weight loss, colour and firmness of avocado stored at 20°C Control, (H>) 5%; (-4-) 25% (-0-) 1.6% and (-*-) 0.6% (w/w) sunflower oil emulsions. 175 Table 34. Effect of oil emulsions on the storage life of Hass avocados stored at 20°C Treatment Time to soften (days) Uncoated 10.25c Sunflower oil emulsion 5% (w/w) nd 2.5% (w/w) 26.13a 1.6% (w/w) 16.0bc 0.6% (w/w) 13.8bc Emulsions were applied as dip. nd = in excess of 26 days. Fruit failed to soften during the duration of the experiment. Values are the means of 6 replicates of 20 fruits. Different letters indicate significant difference at p = 0.05 by DMRT. Summary The desirable colour for Hass avocado when eating ripe is puiple/black (score 1); it is therefore important that the treatments allow normal colour development. Delaying softening is necessary to extend the storage life of avocado and this was successfully achieved by the emulsion containing 2.5% (w/w) sunflower oil. This emulsion is therefore a potential treatment for the storage of avocado at 20°C. 176 4.4.2.3 Nashi Coconut, marakot, sunflower oils and emulsion containing 3.3, 5 and 10% sunflower oil were tested on green Nashi stored at 0° and 20°C. Six drops of oil were smoothly spread on each fruit while the emulsions were applied as dip. Untreated Nashi started to yellow after 6 days storage at 20°C, and were fully yellow by the 18th day (Figure 18). The storage life of Nashi at 20°C, expressed as the time to develop a full yellow colour (score 5), was markedly (p = 0.05) extended by the application of oils and oil emulsions (Figure 18). An average marketable life of 33 days was obtained for treated fruit compared to 18 days for the untreated fruit. i After 11 months storage at 0°C and 9 days at 20°C, untreated fruits were fully yellow (score 5) while coated fruits were at score 2 (25% yellow). Plate 11 and 12 show the effect of oils and oil emulsions on Nashi stored at 20° and 0°C. Weight loss in uncoated Nashi was 4.13% after 33 days of storage at 20°C. Coating Nashi had little effect on weight loss (Figure 18) except for sunflower oil which significantly (p = 0.05) reduced weight loss to 3.2%. The effects of oil coatings on ripening of Nashi stored at 20°C was assessed by determining the change in firmness, total soluble solids, titratable acidity and pH (Figure 19). Firmness value decreased from an initial 4.15kg to 2.42kg in the untreated fruit and to an average 3.6kg in the treated fruit after 19 days storage at 20°C (Figure 19). Marakot and sunflower oils, as well as emulsions containing 5 and 10% (w/w) sunflower oil were more effective in delaying the rate of softening by 31.5% compared to 16% by coconut oil and the emulsion containing 3.33% (w/w) sunflower oil. 177 6 a> o o CO ra o o O CO CO o 03 CD Storage time (days) Figure 18. Effect of oils and oil-based emulsions on colour changes and weight loss in Nashi stored at 20°C. (-»-) Untreated; (-f>) Marakot oil; (4-) Sunflower oil; (0-) Coconut oil; ( -&) 10%; (^) 5% and (o-) 3.33% (w/w) sunflower oil emulsions. Colour score: 1 = green; 2 = 25% yellow; 3 = 50% yellow; 4 = 75% yellow and 5 = fully yellow. Each data point is the mean of 6 replicates of 20 fruits. Vertical bars represent standard errors. 178 Plate 11. Nashi coated with oils and oil-based emulsions after 26 days of siuiagc ai au v^. CO: coconut oil; MKT: marakot oil; SN: sunflower oil; QXDO: 10 /o (w/w), QXD1: 5% (w/w) and QXD2: 3.33% (w/w) sunflower oil emulsions. 179 Control QXD1 Nashi stored for 47 weeks at 0°C + 9 days at 20°C Plate 12. Effect of oil-based emulsions on (a) scald and (b) core and flesh browning of Nashi after 47 weeks storage at 0°C + 9 days at 20°C. QXDi : 5% (w/w) and QXD2: 3.33% (w/w) sunflower oil emulsions. 180 Storage time (days) Figure 19. Changes in firmness, total soluble solids, titratable acidity and pH of Nashi at 20°C. (■*-) Untreated; (-o) Marakot oil; (+) Sunflower oil; (-0-) Coconut oil; (■*■) 10%; (^r) 5% and (-&) 333% (w/w) sunflower oil emulsions. Each data point is the mean of 6 replicates of 20 fruits. Vertical bars represent standard error 181 The total soluble solids and the pH of uncoated Nashi increased from 10.77 °Brix and 3.98, respectively, to 12 °Brix and 4.14, respectively, after 19 days storage at 20°C. These changes were delayed by the treatments (Figure 19) although not to any great extent. Acidity, expressed as malic acid equivalents, decreased with storage but was higher in the untreated fruit than in most of the treated fruit (Figure 19). Sensory evaluation of the fruits after 33 days storage at 20°C indicated that treated Nashi were crispier and juicer compared to the control (Table 35). Off-flavour was strong in fruit coated with Marakot as well as those dipped in 5 and 10% sunflower oil emulsions. There was a greater preference for untreated Nashi and those coated with t sunflower oil, coconut oil or dipped in 3.33% oil emulsion (Table 35). Lenticel injury was the only problem encountered during storage and was observed on fruit dipped in the emulsion containing 10% (w/w) sunflower oil. The injury took the form of pronounced black spotting of the lenticels. 182 Table 35. Sensory attributes of Nashi after 33 days storage at 20°C i Treatment Texture Off-flavour Overall acceptability Untreated 2.02b 3.94bc 6.29a Sunflower oil 4.21a 5.44ab 5.14ab Marakot oil 4.09a 6.02a 4.13bc Coconut oil 4.60a 3.05c 5.75ab Sunflower emulsion 10% (w/w) 4.05a 6.70a 3.56c 5% (w/w) 4.09a 6.44a 4.13bc 3.33% (w/w) 4.40a 3.96bc 6.12a Oils were applied 6 drops/fruit while emulsions were applied as a 10s dip. 1. Overall acceptability score: 9 = liked extremely and 1 = dislike extremely. Each value is the mean of 6 replicates of 20 fruits using 5 panelists. Values followed by different letters within a column are significantly different at p = 0.05 by DMRT. 183 4.4.2.4 Apple Marakot, sunflower oils and emulsions containing 3.3, 5 and 10% sunflower oil were tested on green Granny Smith apples stored at 0° and 20°C. Six drops of oil were also smoothly spread on each fruit while the emulsions were applied as dip. Application of oils and oil emulsions significantly (p = 0.05) delayed yellowing in Granny Smith apples stored at 20°C (Table 36). The greenness of stored apple was measured using a-value in the CIE Lab system (where a negative a-value correlated to greenness). The a-value in the untreated apple was higher (p=0.05) than that of the treated apples after 7 weeks storage at 20°C (Table 36). This indicates retention of green colour in the i treated fruit. Firmness decreased in both the treated and untreated apples during storage. It decreased from an initial value of 5.72kg to 4.59kg in the untreated fruit. Treated apples however remained firmer with an average value of 5.16kg, with no difference between the coatings. Application of oils and oil-based emulsions had tittle effect on the changes in pH, acidity and TSS of apples (Table 36). Weight loss was 2.6% in untreated Granny Smith apples after 7 weeks storage at 20°C (Table 36). This was significantly reduced (p = 0.05) by all coatings, except the 10% oil emulsion, which increased weight loss to 3.06%. This high concentration of oil emulsion also caused injury on the apple in the form of black spotting of the skin. 184 Table 36. Effect of oils and oil emulsions on quality attributes and chemical composition of Granny Smith apples after 7 weeks storage at 20°C. Treatment Firmness Ground colour Weight loss TSS Acidity pH (kg) (a-value)1 (%) (°Brix) (meq/lOOg) Initial value 5.72ab -21.4a 11a 0.56a 3.36a Uncoated 4.59c -9.16c 2.6b 12.75bc 0.39b 3.48b Marakot 5.26b -19.44b 1.98a 13.0c 0.37b 3.47b Sunflower 5.1 lbc -19.14b 1.76a 13.0c 0.41b 3.46b Sunflower emulsion 10% (w/w) 5.24b nd 3.06c 12.75bc 0.40b 3.46b 5% (w/w) 5.19b -19.31b 1.91a 13.0c 0.39b 3.49b 3.33%(w/w) 5.01bc -18.01b 1.99a 12.75bc 0.39b 3.49b Oils were applied as 6 drops/fruit while emulsions were applied as 10 s dips. 1, a more negative value of "a" signifies more green colour. Each value is the mean of 6 replicates of 20 fruits. Values followed by different letters within a column are significantly different at p = 0.05 by DMRT. nd: not determined due to development of unacceptable black injury spotting on the skin. 185 Yellowing was not observed in both the treated and untreated Granny Smith apples after 20 weeks storage at 0°C, although the incidence of superficial scald was observed on the untreated fruit after 9 days at 20°C. This accounted for the low value given by panelists during visual assessment of the whole fruit. Sensory evaluation further showed all treated fruits to be crispier than the untreated apple, with sunflower oil and the emulsions containing 5 and 10% (w/w) sunflower oil having the highest ratings. Off-flavours, however, developed to an unacceptable level (score < 4) in fruit coated with the high concentrations of the emulsion (5 and 10%), subsequently affecting the overall acceptability of the fruit (Table 37). Although the application of sunflower or maralcot oils directly to the skin of the fruit or dipping the fruit in a 3.33% (w/w) sunflower oil emulsion had no significant (p = 0.05) effect on acceptability, they were preferred in terms of visual assessment and therefore recommended as coatings for Granny Smith apples intended for storage at both 0° and 20°C. 186 Expected page number is not in the original print copy. Table 37. Sensory attributes of Granny Smith apples after 20 weeks storage at 0°C and 7 days at 20°C Treatment Visual Texture Off-flavour Overall acceptability assessment1 Untreated 1.0b 2.3bc 4.3a 5.40ab Sunflower oil 4.2a 4.10a 5.8a 6.82a Marakot oil 4.4a 3.18ab 4.76a 5.04ab • Sunflower emulsion 10% (w/w) 2.2b 4.09a 1.76b 3.46c 5% (w/w) 4.2a 4.26a 3.22ab 3.24c 3.33% (w/w) 4.2a 3.68ab 5.92a 5.40ab Oils were applied as 6 drops/fruit while emulsions were applied as 10 s dip. 1. Visual assessment was done using whole fruit and rated from 1 = disliked extremely to 5 = like extremely. Texture rating 5 = very crispy to 1 = soft Off-flavour: 1 = no off-flavour to 7 = strong off-flavour. Overall acceptability score: 9 = liked extremely and 1 = dislike extremely. Each value is the mean of 6 replicates of 20 fruits. Values followed by different letters within a column are significantly different at p = 0.05 by DMRT. 4.4.3 Summary Storage life of the fruit was extended mainly through delayed ripening. The oils and oil emulsions used in this experimental work delayed ripening of avocado, Nashi and apple by retarding the loss of green colour and loss of firmness. Yellowing in cucumber was also delayed. 188 The extent to which the emulsions delayed ripening was dependent on concentration. Higher concentrations may have resulted in a modified atmosphere, containing a higher concentration of carbon dioxide and a lower concentration of oxygen, therefore a greater retention of colour and firmness. However, there appeared to be an adverse effect on the normal metabolism of the fruit. This resulted in injury on the skin of apples and Nashi dipped in 10% (w/w) sunflower emulsion, off-flavours in apples and Nashi dipped in 5 and 10% (w/w) sunflower emulsion whilst avocados dipped in 5% (w/w) sunflower emulsion failed to ripen. 4.5 Coating morphology 4.5.1 Introduction Previous experiments showed that loss of quality was delayed by application of oils and oil emulsions on fruits. This study was carried out to examine the appearance and distribution of the coatings on the surface of Granny Smith apples under scanning electron microscope. In conventional scanning electron microscopy, the image is formed by the detection of "secondary" electrons". These are electrons which are ejected from atoms in the sample as a result of electron bombardment and they have relatively low energy. Therefore only those electrons ejected from atoms in the surface layers of the sample eg the gold coating, are detected and the resulting image has a high topographical information content with good resolution. The trajectories of these low energy electrons are deflected towards the detector by surrounding it with a "cage" to which a positive voltage is applied. If the voltage on the cage is reduced, then only higher energy electrons known as "backscattered electrons" with straight trajectories are detected. These electrons originate from the beam itself and have a much higher energy than the 189 secondary electrons. As a result, the information provided by these electrons may relate to features of the sample at a greater depth than that provided by secondary electrons. 4.5.2 Result Scanning electro microscopy was carried out on the skin of uncoated Granny Smith apples and apples coated with sunflower oil or emulsion containing 3.33% (w/w) sunflower oil. A polysaccharide coating made from sweet potato starch was also studied as a comparison. Electron micrographs of backscatter images of the surface of coated and uncoated fruit are shown in Plate 12 . Scanning electron microscopic (SEM) view of the fruit surface of uncoated apple prior to storage shows the natural cuticular wax as well-oriented with a closely packed platelet structure (Ai). There was no obvious change in the structure of the natural coating on the uncoated apple during storage at 20°C. Under SEM the sunflower oil coating appeared as a continuous uniform film on the fruit surface, while films formed by the 3.33% (w/w) sunflower oil emulsion (C) and sweet potato starch (D) were less even. Partial clogging of the lenticel pores on the fruit skin by the applied coatings was observed (Plate 12 - Bii; Ci,ii; Dii). There was no change in the appearance and structure of the oil or the oil emulsion coatings during storage. However cracking was observed on the sweet potato coating after 2 weeks storage at 20°C. 190 * '• • ' * •* r*- * • 1 It* ■' ► U ,>.i u i. i rn :xl*4i RllSti>fcW^ “ ■ ' psaa® . ■;' i. . tf"h P V,.v V #:-\-v 'V:-.- : : v iwIMr wjilff Plate 13 Electron micrographs of the surface of uncoated apples (A) and those coated with sunflower oil (B), 3.33% (w/w) sunflower emulsion (C) and sweet potato (D) after 0 (i), 4 (ii) and 7 (iii) weeks storage at 20°C. 191 4.5.3 Summary This study demonstrates that unlike polysaccharides, lipid coatings form a continuous film which does not crack during prolonged storage. The film apart from covering the cuticle, also partially and in some cases completely plugged the lenticels. This would restrict the movement of gases across the fruit surface creating a modified atmosphere within the fruit. 4.6 Effect of oil and oil coatings on the permeability of fruit surface to gases and the internal gas composition of Nashi and apple t 4.6.1 Introduction Results from the storage life studies (Section 4.4) showed that application of oils or oil emulsions were promising in extending the storage life of cucumber, avocado, Nashi and apple. It is probable that coatings exert their shelf life extension properties on fruits and vegetables primarily through modifying the permeability of the produce to O2 and CO2. The morphology of the coating during storage (Section 4.5) shows that coating plugs the lenticel pores on the surface of the coating which would result in the restriction of the gas movement This series of experiments were designed to determine the relationship between the coatings and the permeability of Nashi and apple surfaces to oxygen and carbon dioxide. The permeability of fruit skin to oxygen and carbon dioxide was estimated as the rate of uptake or release of each gas by the fruit and its gradient across the fruit surface at 20°C. 192 4.6.2 Results 4.6.2.1 Nashi Gas permeability Untreated Nashi as well as those coated with sunflower, marakot, coconut oils, or dipped in 3.33, 5 or 10% (w/w) sunflower emulsions were held in sealed containers for lh, and the permeability of the fruit surface to O2 and CO2 determined as discussed in Section 3.9 Permeability of uncoated Nashi to O2 was 44.3 (x 10'^ cm h'^ mbar"l). This was significantly reduced (p=0.05) by all coatings lh after application (Table 38). Sunflower, marakot and coconut oils were more effective, reducing the permeability of the fruit surface to O2 to 4.37 (x 10'6 cm h"l mbar"l). The 3.33% (w/w) sunflower emulsion was however the least effective in reducing permeability to oxygen with a permeability of 15.9 (x 10-6 cm h"l mbar" 1). The effect of the emulsions on O2 permeability varied with the concentration of oil present. Permeability in Nashi dipped in 10% (w/w) sunflower emulsion was 6.72 compared to 15.9 in those dipped in 3.33% (w/w) sunflower emulsion (Table 38). The permeability of uncoated Nashi to CO2 was 27.9 (x 10"6 cm h"l mbar'l). Application of the oils and oil emulsions on Nashi fruit significantly reduced (p=0.05) the permeability of the fruit skin to CO2 with the emulsions being more effective (Table 38). As with O2, permeability to CO2 decreased with increasing concentration of oil in the emulsions. Fruits dipped in the emulsion containing 10% (w/w) sunflower oil had a permeability of 0.03 (x 10‘6 cm h'l mbar~l) compared to 1.12 (x 10“6 cm h_l mbar'l) in those dipped in the 3.33% (w/w) sunflower emulsion. Permeability of Nashi coated with sunflower, marakot or coconut oil was within 1.78 - 2.53, with no difference 193 between the oils. Coating therefore reduced the permeability to CO2 by 97% in fruits coated with the emulsions and 92% in those coated with the oils. Overall, in comparison to the oils, the emulsions were more permeable to O2 and less permeable to CO2 (Table 38). Table 38. Effect of oil and oil coatings on the skin permeability to carbon dioxide and oxygen and the internal gas composition of Nashi at 20°C. Gas permeability Internal atmosphere (10"6 cmh‘1 mbar" 1) w Treatment co2 o2 co2 o2 Untreated 27.9a 44.3a 2.97c 14.01a Sunflower oil 2.53b 4.67d 5.61b 1.36b Marakot oil 1.78bc 4.29d 5.75b 2.04b Coconut oil 2.16b 4.16d 5.74b 2.05b Sunflower emulsion 10% (w/w) 0.03d 6.72c 11.75a 2.38b 5% (w/w) 0.27d 7.13c 7.26b 2.52b 3.33% (w/w) 1.12cd 15.9b 6.32b 3.25b Oils were applied as 6 drops/fruit while the emulsions were applied as dip. Each value is the mean of 2 replicates of 3 readings after lh of coating application. Values followed by different letters within a column are significantly different from each other at p = 0.05 by DMRT. Internal Atmosphere One hour after application of the coatings, the CO2 level inside the fruit was 2.97% in the control compared to 5.7% in those coated with sunflower, marakot or coconut oils. 194 Accumulation of CO2 in fruit increased with the concentration of sunflower oil in the emulsion (Table 38). There was however no significant difference (p=0.05) in CO2 concentration in fruits treated with the oils and the oil-emulsions, other than 10% (w/w), in the extent of CO2 accumulation. Internal oxygen concentration in uncoated Nashi fruit was 14.01% after lh at 20°C. It declined sharply (p=0.05) to an average of 2.27% in treated fruits, with no significant difference between the treatments (Table 38). 4.6.2.2 Apple Gas permeability Untreated Granny Smith apples as well as those treated with sunflower or marakot oil, or dipped in 3.33, 5 or 10% (w/w) sunflower emulsions were held in sealed containers for lh, and the permeability of the fruit surface to O2 and CO2 determined. Permeability of the surface of untreated apples to O2 was 17.3 (x 10'^ cm h“l mbar'l). This was significantly reduced (p=0.05) by all treatments lh after application (Table 39). The emulsion containing 10% (w/w) sunflower oil was more effective, reducing permeability of the fruit surface to O2 to 4.05 (x 10"^ cm h"* mbar1). The least effective of the coatings was the emulsion containing 3.33% sunflower oil with fruits dipped in this emulsion having a permeability of 8.93 (x 10“6 cm h~l mbar-!) to O2 (Table 39). Permeability to CO2 was similarly reduced. Untreated apples had a permeability of 1.27 (x 10‘6 cm h‘l mbar-!) to CO2 compared to between 0.10 - 1.13 (10"6 cm h"l mbar‘1) in the treated fruit. The emulsion containing 10% (w/w) sunflower oil was again more 195 effective, decreasing permeability of CO2 to 0.1 (x 10"^ cm h"l mbar"*). Decreasing the concentration of oil in the emulsion increased permeability, with fruit treated with the emulsion containing 3.33% sunflower oil having a permeability of 1.13 (x 10”^ cm h"l mbar'l). Table 39. Effect of oil and oil emulsions on the skin permeability to carbon dioxide and oxygen and the internal atmospheric composition of Granny Smith apples at 20°C. Gas permeability Internal atmosphere (10"6 cm h'l mbar'l) (%) Treatment co2 o2 C02 o2 Untreated 1.27a 17.3a 2.51c 19.5a Marakot oil 0.74c 4.22c 3.24b 15.97c Sunflower oil 0.61b 4.13c 3.05b 16.07c Sunflower emulsion 10% (w/w) 0.10c 4.05c 3.73a I2.87e 5% (w/w) 0.75b 4.33c 3.12b 14.78d 3.33% (w/w) 1.13a 8.93b 2.61c 17.09b Oils were applied as 6 drops/fruit while emulsions were applied as dip. Each value is the mean of 3 replicates of 3 readings after lh of coating application. Values followed by different letters within a column are significantly different from each other at p = 0.05 by DMRT. The internal oxygen concentration of Granny Smith apples was significantly (p=0.05) reduced within lh of coating (Table 28). While the internal oxygen content of the 196 untreated fruit was 19.5%, those coated with morakot and sunflower oils averaged 16.02%. Of the treated apples, those dipped in the 3.33% (w/w) oil emulsion had the highest internal oxygen concentration of 17.09% compared to 12.87 and 14.78% in those coated with 5 and 10% (w/w) sunflower oil emulsions, respectively. Carbon dioxide accumulated significantly (p = 0.05) within treated apples in lh. The carbon dioxide level in the untreated fruit was 2.51%, compared to 3.29% in fruits coated with morakot or sunflower oils as well as those dipped in the emulsions containing 5 or 10% oil. Fruits dipped in emulsion containing 3.33% sunflower oil had similar C02 concentration to that in the intreated fruit. 4.6.3 Summary The results (Tables 38 and 39) show that the surface of untreated Nashi was about twice as permeable to oxygen as it was to carbon dioxide, while Granny Smith apples was 14 times more permeable to oxygen than to carbon dioxide. This suggests that the surface of the fruit is differentially permeable to gases. Burg and Burg (1965) reported a higher diffusivity for oxygen in air than for carbon dioxide but not as high as obtained in these experiment. Coating the fruit reduced the permeability to oxygen and carbon dioxide by 84.6% and 95% respectively in Nashi and 70.3 and 47.5% in apple. The reduction in permeability of the apple skin to oxygen and carbon dioxide was smaller compared to that obtained in Nashi. The reduction in permeability of the fruit surface to the respiratory gases, particularly oxygen, is important with respect to the production of the modified atmosphere environment in the tissue attributed to coatings. Limiting the amount of oxygen in the tissue would limit the amount of oxygen available for respiration which ultimately affects the respiration rate. 197 4.7 Effect of oils and oil emulsions on respiration and internal gas composition of coated fruits. 4.7.1 Introduction The storage life of apples, avocado, cucumbers and Nashi were increased by applying oils and oil emulsions to the fruit. These treatments decreased the permeability of the fruit to oxygen and carbon dioxide and resulted in a changed internal atmosphere. This study aimed at determining the extent of the atmosphere modification created by the coatings and the effect on the respiration rates of avocado, Nashi and apple. 4.7.2 Result 4.7.2.1 Avocado Untreated avocados and those dipped in the 2.5% (w/w) sunflower emulsion were held in respiration jars at 20°C and the respiration rate and internal concentrations of oxygen and carbon dioxide were measured at regular intervals. Avocados had a climacteric respiration pattern with the respiration rate ranging from 29.5 to 112.9 mL C02/kg.hr in the untreated fruit and 22.4 - 48.6 in those dipped in the 2.5% oil emulsion (Figure 20). The respiration rate was markedly reduced by coating the fruit. The climacteric peak was reduced by approximately 50% (Figure 20). 198 120 Storage time (days) Figure 20. Effect of oil emulsion coating on respiration rate and internal gas composition of avocados stored at 20°C (hs ) Uncoated and (-o-) 2.5% (w/w) sunflower oil emulsion. Data points are means of 6 replicates of 20 fruits. Vertical bars represent standard error 199 Internal oxygen concentration declined rapidly from 10% to 3.4% and 2% in untreated and treated avocados, respectively, during the first 5 days storage at 20°C (Figure 20). The decline was greater in the coated fruit. Carbon dioxide on the other hand increased with storage, with a higher concentration in the treated fruit (Figure 20) although there was no significant difference between the treated and untreated fruit until after 12 days of storage at 20°C. The internal CO2 content at this time was 11% in uncoated avocado and 14.9% in the coated fruit. 4.7.2.2 Nashi Nashi fruits were coated with marakot, sunflower or coconut oil or dipped in emulsions containing 3.33, 5 or 10% (w/w) sunflower oil. Fruits were held in respiration jars and respiration rate and internal composition of oxygen and carbon dioxide were measured at regular intervals. The respiration rate of Nashi at 20°C decreased with storage time (Figure 21). The respiration rate of the untreated fruit was 42.9 mL C02/kg.hr at the beginning of the experiment and declined to 17.8 mL CC>2/kg.hr after 11 days. In general, coatings significandy reduced the respiration rate, with the oils being the most effective during the first 7 days of storage. There was however no difference between the respiration rate of fruits coated with sunflower, marakot, or coconut oil, or those dipped in 5 or 10% (w/w) sunflower emulsion by day 11. 200 Figure 21. Respiration Each (-»-) ( -A Respiration rate (mL CC^/kQ-hO t ) 50 Untreated; 10%, data (- point rate a -) 5% (-t>) of is the Nashi and Marakot mean (-&) stored 3.33% of oil; 3 201 at replicates Storage (-f 20°C. (w/w) ) Sunflower sunflower of time 3 fruits. oil; (days) oil (0-) emulsions. Coconut oil; Figure 22 shows the changes in the internal gas compositions of Nashi during storage. Fruit had oxygen concentration close to that of air at the beginning of the experiment. There was not much change in the concentrations of O2 and CO2 of the untreated fruit after 19 days of storage at 20°C. Treated fruit on the other hand developed low oxygen levels (2 - 4%) and high carbon dioxide levels (6.6 - 13.9%) within 9 days of storage compared to 16.54 and 2.97%, respectively in the untreated fruit (Figure 22). The oxygen levels remained at these low levels for the remainder of the storage period with no difference between the coatings. The accumulation of carbon dioxide continued in fruits dipped in the emulsions but declined in those coated with the oils after 19 days storage. There was, however, no significant difference between the effect of the oils and the lower concentrations of the emulsions (3.33' and 5%) on the accumulation of carbon dioxide by the end of storage. Carbon dioxide levels in fruits dipped in 10% (w/w) sunflower emulsion was significantly higher (p = 0.05) than in the untreated and other treated fruits (Figure 22). The carbon dioxide level in Nashi dipped in the 10% (w/w) sunflower emulsion was 18.5% compared to an average of 8.6% in Nashi coated with the other coatings after 19 days of storage. The internal ethylene level of treated and untreated fruit declined throughout storage at 20°C (Figure 22). Internal ethylene of the untreated fruit rapidly declined from an initial 3.2 p.L/L to 0.12 p.L/L after 9 days storage at 20°C. The low level was subsequently maintained through the storage period. A decline to an average of 1.07 pL/L was obtained in the treated fruits after 9 days, with no significant difference (p=0.05) between the coatings. The ethylene concentration continued to slowly decrease in fruits coated with sunflower, marakot or coconut oil and was 1.05 pL/L after 19 days of storage. In contrary, ethylene started to increase in fruits dipped in some of the oil emulsions. 202 20 * 15 5 1C 15 20 Storage time (days) Figure 22. Effect of oils and oil emulsions on the internal oxygen, carbon dioxide and ethylene composition of Nashi at 20°C. (s ) Untreated; (o) Marakot oil; (+) Sunflower oil; (-O) Coconut oil; (-*-) 10%; (-*•) 5% and (&) 333% (w/w) sunflower oil emulsions. Each data point is the mean of 6 replicates of 20 fruits. Vertical bars represent standard error. 203 Although there was no difference between fruits dipped in the emulsion containing 3.33% sunflower oil and those coated with sunflower, marakot or coconut oil, the ethylene level in Nashi dipped in the 5 and 10% (w/w) oil emulsions was 2.4 and 6.6 pL/L, respectively, after 19 days of storage. 4.7.2.3 Apple Sunflower or marakot oils were applied directly to the skin of Granny Smith apples while fruits were dipped in emulsions containing 3.33, 5 or 10% (w/w) sunflower oil. Fruits were held in respiration jars and respiration rates and internal composition of oxygen and carbon dioxide measured at regular intervals. Apples had a climacteric respiration pattern with a minimum rate of 35 mL C02/kg.hr, rising to a maximum of 38.1 mL CC>2/kg.hr after 30 days then declined slightly. The respiration rate started to gradually increase with a slight peak at day 30 followed by a decline to 34.4 mL CC>2/kg.hr after 38 days of storage (Figure 23). A similar trend was observed for all treated fruit although respiratory activity was significantly (p=0.05) suppressed by all treatments. A greater reduction was obtained in apples coated with sunflower or marakot oils or dipped in 5% or 10% oil emulsions with an average respiration rate of 26.1 mL COz/kg.hr compared to 32.9 mL CC>2/kg.hr obtained in those dipped in 3.33% (w/w) sunflower emulsion. 204 40 CD OtJ 35 c= O 30 cd cx CO CD 25 oc 20 —j > •, » i ' i 10 20 30 40 Storage time (days) Figure 23. Respiration rate of Granny Smith apple at 20°C. ( o) Untreated; (-o) Marakot oil; (<^) Sunflower oil; (O) 10%; (nAr) 5% and ) 3.33% (w/w) sunflower oil emulsions. Each data point is the mean of 3 replicates of 3 fruits. Vertical bars represent standard error. 205 Figure 24 shows the changes in the internal gas composition of Granny Smith apple during storage at 20°C. Apples had oxygen levels close to air at the beginning of the experiment. This was reduced to 17.1% in the untreated fruit within 3 weeks of storage and gradually to 11.2% by the end of the 7 week of storage (Figure 24). Coating significantly (p=0.05) reduced the internal oxygen concentration. After 3 weeks apples coated with marakot or sunflower oil or dipped in 5% oil emulsion had an oxygen level of 4.8% while those dipped in 10% (w/w) sunflower oil emulsion had 2.2%. The oxygen levels remained at these levels for the remainder of storage. Internal carbon dioxide levels of both treated and untreated apples increased during f storage (Figure 24). There was an increase in the carbon dioxide level from an initial 2.5% to 5.3% in untreated apple after 5 weeks of storage, followed by a decline to 4.6% over the following 2 weeks. Marakot and sunflower oils had little effect on the accumulation of carbon dioxide throughout the storage period. There was, however, a significant increase (p=0.05) in the level of carbon dioxide in apples dipped in the emulsions. The carbon dioxide levels were 10.7% in those dipped in 10% (w/w) oil emulsion and 9.19% in those coated with 3.33 and 5% (w/w) sunflower emulsions after 5 weeks of storage. This gradually fell to 8.8 and 8.6%, respectively, after 7 weeks of storage. Ethylene was very low at the beginning of storage and could not be detected by the GC at the start of storage (Figure 24). Ethylene increased to 133 \\LfL by week 3 in untreated apples and remained at 315.1 |iL/L until the end of the experiment. Apples coated with sunflower oil or dipped in 10% (w/w) sunflower emulsion had a similar ethylene level (133 ftlVL) to the untreated apples after 3 weeks storage, while the ethylene level in apples coated with marakot, or those dipped in 3.33 or 5% (w/w) 206 20 "7 800 ^ 600 _ 400 0 1 2 3 4 5 6 7 Storege time (weeks) Figure 24. Effect of oils and oil emulsions on the internal oxygen, carbon dioxide and ethylene composition of Granny Smith apple at 20°C. (-» ) Untreated; (-o-) Marakot oil; (+) Sunflower oil; (O) 10%; (■*■) 5% and (■&) 333% (w/w) sunflower oil emulsions. Each data point is the mean of 6 replicates of 20 fruits. Vertical bars represent standard error. 207 sunflower emulsion was 307.4 |iL/L. Internal ethylene was significantly higher (p=0.05) in treated fruits compared to the untreated fruit after 7 weeks of storage, with apples coated with marakot having an ethylene level of 815.9 |iL/L and those dipped in emulsions containing 3.33 and 10% (w/w) sunflower oil having the lowest concentration of 512.6JJ.L/L. 4.7.3 Summary It is established from this experiment that the coatings modify the internal atmosphere of the coated fruit. The effect on oxygen was greater than that on carbon dioxide in both coated Nashi and Granny Smith apple. The effect of the coatings on the respiration rate was not as great in apples and Nashi compared to the effect on avocado. A low concentration (2.5% w/w ) of the sunflower oil emulsion resulted in a significant reduction in the respiration rate of avocado. 208 4.8 Effect of coating on physiological disorder development and internal atmosphere of Nashi and apples stored at 0°C. 4.8.1 Introduction Low temperature storage is used for the storage of Nashi and apples. Both fruits are subjected to physiological disorders after periods of storage at low temperatures. Disorders associated with Nashi and apples include superficial scald, core and flesh browning and senescence breakdown. This experiment was designed to examine the effect of oil and oil emulsions oh the development of physiological disorders and the relationship between incidence of disorders and the internal atmosphere composition of Nashi and apples. f 4.8.2 Result 4.8.2.1 Nashi Physiological disorders Nashi fruits coated with marakot, sunflower or coconut oil or dipped in emulsions containing 3.33, 5 or 10% (w/w) sunflower oil were stored at 0°C. Fruits were removed from 0°C after 47 weeks of storage and assessed for physiological disorders after 9 days at 20°C. Physiological disorders observed in untreated Nashi were scald, core and flesh browning. The scald, characterised by a bronze appearance, on the skin developed on 68.5% of the untreated fruit (Table 40). The occurrence of the disorder was completely prevented by all the treatments (Table 40). 209 Table 40. Effect of oils and oil emulsions on the development of physiological disorders in Nashi after 11 months storage at 0°C. Treatment Scald Core browning Flesh browning Untreated 3.24a (68.5) 4.46a (85.4) 4.52a (92) Sunflower oil 1.0b 2.0bc (31.6) 1.0b Marakot oil 1.0b 2.6bc (76.9) 1.3b (18.7) Coconut oil 1.0b 2.7bc (64.5) 1.0b Sunflower emulsion 10% (w/w) 1.0b 1.5c (34.5) 1.0b 5% (w/w) 1.0b 2.2bc (35.7) 1.0b 3.33% (w/w) 1.0b 2.87b (56.7) 1.0b Oil was applied as 6 drops/fruit while the emulsions were applied by dipping. Severity of disorders were assessed using a 1 - 5 score scale, where 1 = no disorder, 2 = slight, 3 = moderate, 4 = severe and 5 = very severe. Values in parentheses are % affected fruits. Each value is the mean score of 6 replicates of 20 fruits. Values followed by different letters within a column are significantly different at p = 0.05 by DMRT. 210 Core browning was observed in 85.4% of the untreated fruit, with a severity score of 4.5. This was significantly reduced (p=0.05) by all the other treatments. The mean value was 2.3 with 48.7% of fruit being affected (Table 40). Although the severity of core browning in the treated fruits were similar, sunflower oil and emulsions containing 5 and 10% (w/w) sunflower had the least affected fruit (31.6, 34.5 and 35.7% respectively.). About 92% of the untreated Nashi were affected by flesh browning with a severity score of 4.5 (Table 40). All treatments other than marakot completely prevented the occurrence of this disorder. Only 18.7% of fruits coated with marakot were however affected. Plate 13 shows the effect of coatings on physiological disorders of Nashi. Internal gas composition Figure 25 presents the changes in the internal gas compositions of Nashi during storage at 0°C and subsequent removal to 20°C. Application of the oils and oil emulsions modified the internal gas composition of Nashi with the effect of treatment type being greater on carbon dioxide than oxygen and ethylene. Internal oxygen of the uncoated fruit declined from an initial 16.5% to 2.2% after 11 months storage at 0°C. The oxygen level increased after fruits were removed to 20°C and was 9.1% after 2 weeks. A similar decline in oxygen to 0.92% was observed in all treated fruits after 11 months storage at 0°C, but only slight increase in the oxygen level occurred when fruits were held at 20°C. 211 Expected page number is not in the original print copy. 20 0 5 10 15 v------0CC------20°C - Storage time (months) Figure 25. Effect of oils and oil emulions on the internal oxygen, carbon dioxide and ethylene composition of Nashi during storage at 0°C and subsequent holding at 20°C. (-E-) Control; k>) Marakot; (-4) Sunflower; (-<>-) Coconut oils; O ) 10%; (-A-) 5%; and Hi) 3.33% (w/w) sunflower oil emulsions. Each data point is the mean of 2 x 3 replicates of 20 fruits. Vertical bars represent standard error. 213 The carbon dioxide level remained low (0.3 - 0.8%) in the untreated fruit throughout storage at 0°C but increased to 8.66% after 2 weeks at 20°C. Carbon dioxide significantly increased (p=0.05) in all treated fruit during storage at 0°C, and was between 6.2 - 13.2% after 11 months. Although carbon dioxide level increased during storage at 20°C, the effect was greater on fruits dipped in the 10% (w/w) sunflower emulsion. The carbon dioxide level was 16.6% compared to 9.7% for the other treatments after 22 weeks at 20°C. Ethylene concentrations remained low in both treated and untreated Nashi during storage at 0°C (Figure 25). There was a rapid increase from 0.34 to 13.1 (iL/L in untreated fruit t after removal to 20°C. A similar upsurge to 7.5 and 9.51 pIVL was observed in fruits coated with marakot or dipped in 5% (w/w) sunflower emulsion respectively. Ethylene levels in fruits coated with sunflower or coconut oil, or dipped in emulsions containing 3.33 or 10% (w/w) sunflower oil however remained low (0.35 - 1.8 pIV after 1 week storage at 20°C (Figure 25). 214 4.8.2.2 Apple Physiological disorders Granny Smith apples coated with marakot or sunflower oil or dipped in emulsions containing 3.33, 5 or 10% (w/w) sunflower oil were stored at 0°C. Fruits were removed from 0°C after 20 weeks storage and assessed for physiological disorders after 7 days at 20°C. Superficial scald developed on untreated apples with a severity score of 4.1 and 100% of the fruit were affected. The occurrence of superficial scald was markedly reduced (p=0.05) and in some cases completely prevented by the coatings (Table 41). Sunflower and marakot oils as well as the emulsion containing 3.33% (w/w) sunflower oil reduced the severity of superficial scald to 1.3, 1.8 and 1'.7, respectively, with 18.3, 22 and 64.5% of fruit affected. Superficial scald was completely prevented in apples dipped in 5 or 10% (w/w) sunflower emulsions (Table 41). Plate shows the effect of the oils and oil-based emulsions on the development of superficial scald on apples stored at 0°C. Core flush was observed in both treated and untreated fruit. This appeared as 'pink blushes' on the flesh of the fruit when cut equatorially. The incidence of core flush was lower in fruits coated with sunflower oil and those dipped in 3.33 or 5% (w/w) sunflower oil emulsions (Table 41). 215 Table 41. Effect of oils and oil emulsions on the development of physiological disorders in Granny Smith apple after 20 weeks storage at 0°C Treatment Superficial scald Core flush Untreated 4.12(100) 1.19(68.3) Sunflower oil 1.28(18.3) 1.4 (64.3) Marakot oil 1.82(21.7) 2.76 (75.2) Sunflower emulsion 10% (w/w) 1.0 (0) 2.76 (69.2) 5% (w/w) 1.0 (0) 2.42 (55.3) 3.33% (w/w) 1.69 (55.5) 1.41 (68.1) LSD (0.05) 0.16f 0.48 Oil was applied as 6 drops/fruit while emulsions were applied by dipping. Severity of disorders were assessed using a 1 - 5 score scale, where 1 = no disorder, 2 = slight, 3 = moderate, 4 = severe and 5 = very severe. Values in parentheses are % affected fruits. Each value is the mean score of 6 replicates of 20 fruits. Values followed by different letters within a column are significantly different at p = 0.05 by DMRT. 216 Plate 14. Effect of oils and oil-based emulsions on superficial scald of Granny Smith apples after 20 weeks of storage at 0°C (a) + 1 week and (b) + 3 weeks at 20°C. MKT: marakotoil; SN: sunflower oil; QXD0: 10% (w/w), QXDi: 5% (w/w) and QXD2: 3.33% (w/w) sunflower oil emulsions. Internal gas composition Changes in the internal gas composition of apples during storage at 0°C and after holding at 20°C are shown in Figure 26. Internal oxygen content of untreated apple declined from an initial value of 19.5% to 13% within 9 weeks of storage at 0°C. The oxygen level gradually rose to 16.1% by the end of the 20 weeks storage period. Moving the fruit from 0° to 20°C did not have much effect on the oxygen level of the untreated fruit with the oxygen level remaining at 16.0% after 2 weeks at 20°C. Oxygen content was drastically reduced (p=0.05) by coatings throughout the storage period. A low oxygen level of 5.1% was observed in apples coated with sunflower oil and those dipped in emulsions containing 5 and 10% (w/w) sunflower oil after 20 weeks storage at 0°C, t while oxygen levels in apples coated with marakot or dipped in 3.33% (w/w) sunflower emulsion were 12.9 and 15%, respectively. A sharp decline to 2.3% was however observed in the latter after removal to 20°C. The oxygen level in all treated fruit was between 1.9 - 4.5% after 2 weeks at 20°C compared to 16.03% in the untreated fruit. The carbon dioxide level in the untreated fruit remained low (1.13%) throughout storage at 0°C (Figure 26). There was an increase after removal to 20°C and was at 5.5% after 2 weeks. Carbon dioxide was significantly higher (p=0.05) in all treated fruits during storage at 0°C. The emulsion containing 10% (w/w) sunflower oil was more effective in altering the carbon dioxide concentration. This treatment had a carbon dioxide level of 12.4%, while marakot and 3.33% (w/w) sunflower emulsion were the least effective with an average carbon dioxide level of 7% after 20 weeks of storage. Carbon dioxide increased in all treated apples upon removal to 20°C and was between 15 - 16% in apples coated with sunflower and those dipped in 5 or 10% (w/w) sunflower emulsion after 2 weeks and an average of 9.8% in those coated with marakot or dipped in 3.33% (w/w) sunflower emulsion. 218 20 Storage time (weeks) Figure 26. Effect of oils and oil emulsions on the internal oxygen, carbon dioxide and ethylene compositions of Granny Smith apple at 0°C and subsequent holding at 20°C. ('■') Untreated; (-o) Marakot oil; (-4) Sunflower oil; ( 219 Ethylene concentration remained low (16.3 |lL/L) in the untreated fruit throughout storage at 0°C and increased to 88.1 jiL/L after 2 weeks at 20°C. There was a gradual increase in ethylene level in all the treated fruit during storage at 0°C (Figure 26) but only those coated with sunflower or marakot oil were significantly different from the untreated apples with an ethylene content of 155.01 \\LfL compared to 15.1 |iL/L in the untreated apples after 20 weeks of storage at 0°C. A sharp upsurge was however observed in apples when removed to 20°C. Apples dipped in 3.33% (w/w) sunflower emulsion and those coated with marakot oil had an ethylene concentration of 540.2 |iL/L compared to a concentration of 119.5 (iL/L in the other treated fruits. 220 4.9 Effect of coatings on a -farnesene and putrescine production in Granny Smith apples. 4.9.1 Introduction The previous experimental work showed that the application of coatings on apples prevented the development of superficial scald and reduced core flush during storage at 0°C. The oxidation of a-famesene has been associated with superficial scald of apples (Huelin and Coggiola, 1970a, b, c). Putrescine is associated with cold stress and has been linked with the development of chilling injuries in some fruit (McDonald and Kushad, 1986; Yuen et al., 1995). This study examines the effect of oils and oil-based emulsions on the level of a-famesene and putrescine in Granny Smith apples during storage at 0°C. 4.9.2 Results A low level (1.8 mg/100cm2 peel) of a-famesene was observed in the epicuticular wax of apples prior to storage. Untreated apples showed a significant increase in a-famesene level shortly after exposure to 0°C (Figure 27), reaching a peak value of 153.3 mg/100cm2 peel after 9 weeks. The level of a-famesene started to decline after 9 weeks and was 125 mg/100cm2 peel at the end of 20 weeks storage. Application of coatings significantly reduced (p = 0.05) the accumulation of a-famesene over the 20 weeks storage period (Figure 27). Sunflower oil was more effective with an a-famesene level of 30.8 mg/100cm2, while 3.33% (w/w) sunflower emulsion was the least effective with a level of 73.4 mg/100cm2 after 20 weeks of storage at 0°C. 221 200 73 150 storage time (weeks) Figure 27. Changes in a-farnesene and putrescine levels of Granny Smith apple stored at 0°C. (■•■) Untreated; (-o-) Marakot oil; (-f) Sunflower oil; ) 10%, (^) 5% and (-£-) 3.33% (w/w) sunflower emulsions. Each data point is the mean of 6 replicates of 5 fruits. Vertical bars represent standard error. 222 The putrescine level was also low (0.2 pg/g) prior to storage. There was an initial upsurge in putrescine production to a level of 6.5 pg/g in the untreated apple after 9 weeks of storage (Figure 27). This was followed by a rapid decline to 1.3 pg/g peel by week 15, after which putrescine concentration remained relatively constant until the end of storage. All treatments delayed accumulation of putrescine with the putrescine level of all treated fruits peaking 5 weeks after the untreated. Sunflower oil was more effective in reducing the level of putrescine with a level of 2.8 pg/g compared to 10.6 p g/g in apples dipped in the 10% (w/w) sunflower emulsion after 15 weeks of storage. The putrescine level fell in all treated apples to the same low level of about 1.0 pg/g peel, as did the untreated fruits after 20 weeks of storage (Figure 27). 4.9.3 Summary The accumulation and decline in the level of putrescine coincided with that of a- famesene in the untreated apples. This was not so in the treated fruits with the production of a-famesene remaining fairly constant during storage at 0°C even though there were rapid changes in the putrescine production. More work should be done to ascertain the relationship between putrescine, a-famesene and low temperature injury of apple. 223 5 DISCUSSION 5.1 Introduction The principal aim of this project was to develop modified atmosphere packaging in the form of lipid-based edible coatings which are relatively simple to apply, safe, cost- effective, but at the same time, easily accessible and affordable to the average farmer in developing countries, as well as in developed countries. This will allow for quality maintenance and postharvest life extension of a range of fruit and vegetables handled under ambient, as well as low temperature conditions. The effect of a number of coatings made from some commercially available oils and fats on quality shelf life extension, skin permeability, internal atmosphere, respiration pattern 4 and the development of physiological disorders of selected fruits and vegetables were examined. Preliminary evaluation of a range of coatings made from various fats and oils, with different chain lengths, fatty acids or degree of unsaturation, on a range of fruits and vegetables showed that lipid coatings were promising in extending the storage life of fruit and vegetables. However, skin coatings that were too concentrated were found to induce anaerobiosis and/or led to failure of some fruits to ripen normally. The major studies involved those lipids which showed the greatest potential. The performance of an oil emulsion was also studied, especially in respect to the effect of the concentration of the oil component. Produce which demonstrated a good response to the lipid coatings and emulsions include fruit such as avocado, lychee, plum, nectarine, banana, apple, Nashi and pear, fruit- vegetable such as cucumber and vegetables such as capsicum and carrot. Coatings were not only ineffective on zucchini, they enhanced senescence and deterioration instead. 224 In addition to shelf life extension of selected fruits and vegetables, a number of coatings were also found to be promising in controlling low temperature physiological disorders such as superficial scald, core and flesh browning in apples and Nashi fruit during cold storage. The other major objective of the research was to elucidate whether the coatings were behaving as a form of modified atmosphere packaging. This was done by examining the effect of the coatings on skin permeability, internal atmosphere, and respiration pattern of some fruits. 4 5.2 Effect of coating on storage life Storage life was assessed as the time for the produce to be just marketable. This was determined on the basis of colour and firmness in plum, banana, apple Nashi, avocado and pear; yellowing in cucumber; browning in lychee and shrivelling in carrot and nectarine. The lipid and lipid-based emulsion coatings used in this study significantly delayed the development of browning in lychee by 4 - 7 days, shrivelling in carrots and capsicums by 9 and up to 18 days respectively, and yellowing in cucumber by up to 30 days. Ripening in Nashi and apple was markedly delayed by 16 and 35 days respectively. The mechanism of ripening is thought to be associated with the breakdown of "organisation resistance" which results in the release or activation of bound enzymes to react with substrates, co-factors, inhibitors and activators (Rhodes, 1980). Ripening is therefore associated with increased enzymic activities (Rhodes, 1980). 225 The colour changes observed in ripening fruit are the result of a number of reactions including the breakdown of chlorophyll and biosynthesis of carotenoids and anthocyanin. Browning of lychee peel has also been associated with the oxidation of polyphenol by polyphenol oxidase (Akamine, 1960a). A feature of these enzyme catalysed reactions is that they are oxygen dependent so limited oxygen availability would affect the rate of reaction. The exclusion of oxygen through CA/MA storage has been observed to reduce the rate of chlorophyll degradation and biosynthesis of yellow and orange colours (carotenoids) and red and blue colours (anthocyanin) in fruits and vegetables, as well as reduce browning in lettuce (Siriphanich and Rader, 1985), mushroom (Murr and Morris, 1974) and snap beans (Buescher and t Henderson, 1977). It is suggested that the delayed colour changes observed in this study may be a result of the same mechanism. The ability of the coatings to create a low oxygen and/or high carbon dioxide environment in the fruit was in fact established in this work. The delayed ripening observed in the fruit and vegetables could also be due to the effect of the MA induced by the coatings on ethylene production and action. Ethylene, a plant hormone which regulates many aspects of growth, development and senescence (Abeles, 1973), is considered to be an inducer of the ripening process in many fruits (Lieberman, 1979). Its synthesis and action are however oxygen dependent (Adams and Yang, 1979; Knee, 1980; Trewavas, 1982). Ethylene is however thought to act by binding to a receptor forming an activated complex which in turn triggers the primary reaction that initiates the chain reaction and finally leads to a wide range of physiological responses including ripening (Trewavas, 1982). Oxygen is required around the binding site for binding to occur, as well as needed for the oxidation 226 of ethylene during action (Beyer, 1975). Carbon dioxide is also known to act as a competitive inhibitor with the ability to displace ethylene from the binding site. The modified atmosphere effects generated by the coatings used in this study could have acted via one or both of the following; 1) The increased level of carbon dioxide within the produce may have inhibited the binding of ethylene to the receptor consequently delaying ethylene action which includes colour loss. 2) The oxygen level though not low enough to affect ethylene production may have inhibited the oxidation of ethylene during its action. In this study, ethylene production in apples and Nashi was observed not to be affected b> the reduced oxygen generated by the coatings, so the latter may account for the delay in 4 colour loss of coated apples and Nashi even though their ethylene concentrations were higher than the uncoated fruits. Coating delayed the rate of softening in all fruits evaluated in this study. Coated Nashi and apple were crispier in comparison to the uncoated fruits based on subjective assessment. Tissue softening is associated with the alteration of polymeric carbohydrates in the middle lamellae and the primary cell walls. The pectic polysaccharides undergo polygalacturonase hydrolysis to form water-soluble pectic substances (Bartley and Knee, 1982). The disintegration of pectic substances and cellulose fibrillar materials observed during the storage of pears (Ben-Arie and Sonego, 1979), mangoes (Roe and Bruemmer, 1981), avocado (Pesis et al., 1978) and apples (Han et al., 1985) indicates the involvement of polygalacturase and/or cellulase in softening of fruit flesh. Wang (1990) reported a 50 - 60% suppression in the activities of cellulase and polygalacturonase during ripening of avocado fruit in 2.5% oxygen compared to those ripened in air. Therefore, the delayed softening observed in coated fruits in this study is most likely the result of reduced activity of enzymes such as polygalacturase or cellulase, that are responsible for the degradation of the cell wall. It is not clear if the reduced enzyme 227 activity is a consequence of reduced oxygen or elevated carbon dioxide but previous work by Johnson (1982) suggests that raised levels of carbon dioxide can improve fruit firmness when associated with oxygen levels below 1%. The hydrophobic nature of the lipid coatings used in this study restricted the movement of water from the fruit to the surrounding environments. This consequently reduced moisture loss in most of the produce evaluated and this is of particular commercial advantage in nectarines, whose shelf life is reduced by shrivelling that results from moisture loss. The antitranspirant property of oils was similarly used to reduce weight loss in banana (Jones el ah, 1978). i The extent to which the emulsion delayed ripening was dependent on the oil concentration. Higher concentrations gave much lower oxygen and much higher carbon dioxide levels resulting in a greater delay in colour changes and softening. The modified internal atmosphere generated by the 5 and 10% (w/w) sunflower oil emulsions, had an adverse effect on the metabolism resulting in injury to the skin of apples and Nashi, off-flavours in apple and Nashi and failure of avocados to ripen. The extended storage life achieved in this study was related to the reduced oxygen and elevated carbon dioxide levels that resulted from the modification effect of the coating. The atmosphere modification was considered to be a result of changes to skin permeability. 5.3 Mechanism of action of coatings The immediate effect of coating on produce is the modification of the internal atmosphere of the produce as a result of changes in the permeability of the surface to gaseous exchange. This was confirmed in Nashi and apple. The application of oils by hand at reduced levels (4-6 drops) resulted in approximately 20 mg of coating on the fruit surface 228 while the sunflower oil emulsions when used as 0.6, 1.6, 2.5, 3.33, 5 or 10% (w/w) oil left between 13-40 mg of coatings. The films formed were invisible except for a slight sheen imparted to the skin of the produce. Scanning Electron Micrograph (SEM), showed that the film formed was continuous with no apparent change in structure during the storage period in comparison to cracking observed in films formed from polysaccharide based coatings. The lipid coatings partially, and in some cases completely, plugged the lenticels. This reduced the movement of gases through the fruit surface. Oxygen within the fruit is therefore used up with no corresponding replacement, as well as carbon dioxide produced « by the fruit being accumulated. Similar observations were made by Banks (1984) and Ben- Yehosua (1985). They proposed that the deposit reduced the rate of movement of oxygen, carbon dioxide and ethylene gases into the fruit via the stomata, lenticels and other pores. In this study, restriction of gas exchange by the lipid coatings created a modified atmospheric condition which was dependent on the combined permeabilities of the lipid films and that of the skin of the fruit. 5.4 Effect of coating on permeability' of fruit surface to gases Application of oils and oil emulsions altered the permeability of the skin of apple and Nashi to oxygen and carbon dioxide. Permeability of the fruit to the gases was observed to be reduced after 1 h of the coating application. The surfaces of both fruits were more permeable to oxygen than to carbon dioxide. Nashi was about twice as permeable to oxygen as it was to carbon dioxide and about 14 times in apple. This is supported by the finding of Burg and Burg (1965) that oxygen has a higher diffusivity in air than carbon dioxide, although not as high as obtained in these experiment. In contrast, Banks (1984) observed bananas as having a greater permeability to carbon dioxide than oxygen and ethylene. This could be due to the high respiration rate of bananas as compared to apple 229 and Nashi, and considering the fact that the gaseous exchange within 1 hr was used in determining the permeability in both studies. The difference in the permeability of the fruit surface to oxygen and carbon dioxide obtained in this study however, suggests that the surface of each fruit is differentially permeable to gases. Coating reduced the permeability to oxygen and carbon dioxide by 84.6% and 95%, respectively, in Nashi and 70.3 and 47.5%, respectively, in apple. The reduction in permeability of the apple skin to oxygen and carbon dioxide was smaller compared to that obtained in Nashi and this could be related ta their metabolic activities. Apple has a lower i activity supported by the slight change in its internal oxygen content after 1 hr of coating application (Table 28). This increased the concentration gradient and reduced the calculated gas permeability. The ratios of the permeability of oxygen to carbon dioxide in the coated fruit was different to that obtained in the control fruits. This suggests that the effect of the coating on the permeability of the skin to oxygen and carbon dioxide would not only be the result of an enhancement of differential permeability which was inherent in the nature of the fruit surface as suggested by Banks (1984), but also includes the differential properties of the coatings themselves. The permeability to oxygen was greater in fruits dipped in the emulsions than those coated with sunflower, marakot and coconut oils. The reverse was the case for carbon dioxide, with the oils having a greater permeability to carbon dioxide than the emulsions. This suggests that carbon dioxide is more soluble in lipid than in water, since the major difference in the two coatings was the water present in the emulsion. This is supported by the report that carbon dioxide is 3 - 8 times more soluble in organic solvents than in water, as well as its total solubility in fats being significantly higher than for the other gases (10 times more than for oxygen) (Mitz, 1979). 230 The overall effect of reduced permeability of the fruit surface to gases was greater on oxygen than carbon dioxide. The oxygen content of uncoated Nashi was 6 times more and the carbon dioxide 2 1/2 times less than in the coated fruit after 19 days of storage at 20°C. Similarly for apple, oxygen was 3 times more and carbon dioxide 1 1/2 times less in the uncoated fruit than in the coated fruit after 7 weeks of storage. So far in this study it has been suggested that the extended storage life achieved by the lipid coatings occurred as a result of the modified atmosphere created. It was established experimentally that coatings reduced the permeability of fruit surface to gaseous exchange 4 modifying the internal gas composition to low oxygen and high carbon dioxide levels. The modified atmosphere effect of the lipid coatings in this study is not only on reducing activity of the enzymes involved in the ripening process but also on reduced sensitivity of the fruit to ethylene, reduced respiration rate and controlled development of physiological disorders. The amount of oxygen available to carry out some important processes is reduced, subsequently reducing the rate of reaction. Reactions affected are the oxygen- dependent processes which include ethylene action, and respiratory metabolism. 5.5 Effect of coating on sensitivity of fruit to ethylene Oxygen is needed in the synthesis of ethylene (Knee, 1980, Yang, 1985). It is expected that the more oxygen available, the more ethylene produced. Subsequently, the ethylene content of the uncoated fruit should be higher than the coated fruit. This was, however, not found in this study, with ethylene levels in coated apples and Nashi being higher than in the uncoated fruit. This could be explained by the fact that ethylene concentration measured in this experiment does not necessarily give the total amount produced, since some of the ethylene produced in the uncoated fruit would have been involved in other 231 processes such as ripening. Although the ethylene content of coated fruits was high there was no subsequent response of ripening. It could therefore be proposed from this study that coating reduces the sensitivity of fruit to ethylene. Reduced sensitivity of the tissue to ethylene observed in this study may be the result of the inhibiting effect of high carbon dioxide levels on ethylene binding to the receptor and/or insufficient level of oxygen necessary for the oxidation of ethylene during action as suggested by Burg and Burg (1967). 5.6 Effect of coating on respiratory metabolism 4 Avocado, apple and Nashi, fruits which exhibit a climacteric pattern of respiration showed a reduced respiratory activity when coated with the lipid coatings. About 50% reduction was observed in avocado, a fruit with a high respiration rate, during the climacteric rise. Reduced respiration rates have also been reported in coated apples (Trout et al., 1953), avocado (Durand et ah, 1984) and banana (Banks, 1984). The rate of respiration of fresh fruits and vegetables has been reported to be markedly affected by low oxygen or high carbon dioxide concentrations. Solomos (1982) suggested that the decrease in respiration rate in response to low oxygen may be the result of a diminution of activity of an oxidase which has a 5 - 6 times lower affinity than cytochrome oxidase, especially when the low oxygen level is sufficient for the activity of cytochrome oxidase. Although C>2 is not directly involved in the reactions of the Krebs cycle, the cycle can only operate in a commodity under aerobic conditions. NAD+ and FAD in the mitochondrion can be regenerated only by the transfer of electrons from NADH and FADH2 to O2 (in the oxidative phosphorylation pathway) with the formation of CO2. The electron-carrier chain is made up of 3 enzyme complexes called NADH-Q reductase, cytochrome reductase and cytochrome oxidase. Cytochrome oxidase is oxygen dependent, 232 catalysing the transfer of electron from reduced cytochrome c to oxygen. A reduced oxygen level will therefore affect its activity. Studies of the effect of high CO2 levels on Krebs cycle intermediates and enzymes showed accumulation of succinic acid due to inhibition of succinic dehydrogenase activity in apples (Hulme, 1956; Knee, 1973; Monning, 1983), pears (Frenkel & Patterson, 1973) and lettuce (Brecht, 1973). Shipway and Bramlage (1973) found that CO2 levels above 6% stimulated malate oxidase and suppressed oxidation of citrate, a-ketoglutarate, succinate, fumarate and pyruvate in mitochondria isolated from apples. Ultrastructural changes (fragmentation, reduction in size and changes in shape of mitochondria), similar to those 1 associated with senescence, was also observed in "Bartlett" pears stored at elevated CO2 (Frenkel & Patterson, 1977). Since the intermediate compounds of the Krebs cycle was beyond the scope of this work, it is not clear if the modified effect of the coating on respiration rate was a consequence of an elevated carbon dioxide level or reduced oxygen level. The effect was however greater on the respiration rate of avocado compared to apple and Nashi. This is due to the normally low respiration rate of apple and Nashi as observed in the uncoated fruits compared to the avocados’ high respiration rate. A very low concentration (2.5% w/w ) of the oil emulsion, however, exerted a great reduction in the respiration rate of avocado which was not concomitant with the reduced oxygen achieved by the coating. Banks (1984) observed a similar phenomena in coated bananas. 233 5.7 Effect of coating on development of physiological disorders Physiological disorders refer to breakdown of tissue that is not caused by either invasion by pathogens or by mechanical damage. Many of these disorders occur as a result of prolonged periods of cool storage and/or exposure to high levels of ethylene, carbon dioxide and low levels of oxygen. Low temperature generally causes a reduction in respiration rate and metabolism. The overall effect may be an imbalance in metabolism, disrupting the production of essential substrates which results in toxic products being accumulated (Wills et al., 1989). Some of these toxic compounds have been associated with the occurrence of some physiological disorders. Most important of these compounds are the oxidative products of a-farnesene. i Lipid coatings used in this study dramatically reduced and in some cases prevented physiological disorders such as superficial scald, core and flesh browning in Nashi and superficial scald and core flush in Granny Smith apples after prolonged storage at 0°C. Little work has been reported on scald of Nashi, which is probably a senescence scald that appears after long-term cold storage (White et ah, 1990). White et al (1990) reported that reducing ethylene in the storage environment could reduce the development of scald and therefore recommended good ventilation to reduce the concentration of ethylene in the storage environment. Results obtained from the internal gas measurement showed that there was no difference in the internal ethylene concentrations of the coated and uncoated Nashi, even though the former did not develop scald. The internal ethylene concentration of the fruit does not necessarily quantify the concentration in the storage atmosphere, especially with an artificial barrier as in the case of the coated fruit. The change in colour of the uncoated Nashi indicates ripening which must have involved ethylene action. It would therefore be reasonable to assume the ethylene concentration in the storage environment of the uncoated 234 fruit to be higher than for the coated produce. The ethylene level around the coated fruit must have been low enough to have prevented scald development on the fruit. Many workers have related the development of superficial scald of apples to the concentration of conjugated trienes (Huelin and Coggiola, 1970a,b,c; Chen et al., 1990). Its labile nature and the fact that it constitutes at least 1% of the ‘Granny Smith’ cuticle oil fraction suggested that it may have a role in the development of superficial scald (Murray et al., 1964). During storage most of the scald appeared after the a-farnesene reduced a maximum, and continued to increase while a-farnesene decreased (Huelin and Coggiola, 1968). This suggested that the production of a-farnesene is only the first stage in the development of scald and that the oxidation products of a-farnesene are the causal agents. The scald-promoting factor appears to be formed by an oxidation process during storage rather than by direct synthesis (Ingle and D’Souza, 1989). Severity of scald development was observed to be proportional to extent of a-farnesene oxidation. Oxidation of a-farnesene produces toxic volatiles (Huelin and Murray, 1966; Huelin and Coggiola, 1970) which accumulate on the apple skin resulting in browning of the skin (Brooks et al., 1919). Oils and oil emulsions used in this study reduced the incidence of superficial scald in Granny Smith apple, a result similar to that obtained by Scott et al (1995) where various vegetable oils reduced scald on Granny Smith apples. Since a-farnesene has been related to the occurrence of superficial scald, its level in coated and uncoated apples was measured during storage at 0°C. 235 In this study, a decline in the a-farnesene level of the uncoated apple skin after 9 weeks storage may have resulted from its oxidation. The level in most coated fruit remained slightly higher than the initial level throughout cold storage. The reduced oxygen and carbon dioxides level in the coated fruit has an inhibiting effect on the production and/or oxidation of a-farnesene subsequently inhibiting scald development. A similar lowering in the degree of a-farnesene accumulation was observed in apples kept in an atmosphere containing 3% oxygen and 5% carbon dioxide (Morozova et al., 1974). Previous experimental work carried out at the University of New South Wales showed that a-farnesene accumulated in the skin of Granny Smith apples during storage at 20°C (Patrick, 1994, unpublished). The a-farnesene level peaked and then started to decline, a trend similar to what was obtained in apples stored at 0°C in this present study. Considering a similar trend in the accumulation of a-farnesene in uncoated Granny Smith apple at 0° and 20°C, with no scald developing at 20°C, low temperature in addition to the oxidation of a-farnesene may have a role in the development of superficial scald. Prolonged storage at low temperature storage could result in freezing of water outside the cells in the fruit tissue. This leads to the collapse of the cell integrity. Earlier reports that scald symptoms begin with oxidative browning products and breakdown of the cytoplasm in hypodermal cells suggest that it is more likely that conjugated trienes enter the cells and attack the tonoplast to cause the subsequent tanning events (Bain, 1956, Bain and Mercer, 1963). 236 A recent report suggest that ethanol vapour can reduce the incidence of superficial scald in Granny Smith apple (Scott et. al., unpublished), the modified atmosphere generated by the coatings used in this study may have increased the ethanol level in the internal atmosphere of the coated fruits. This was evident by off-flavours detected in some of the fruit during the sensory evaluation. The increased ethanol content may have reduced the freezing temperature of the tissue, subsequently preventing tissue breakdown which would have resulted in the development of some of the low temperature physiological disorders. The accumulation of putrescine, a compound closely related to cold hardiness and freeze resistance of plants (Galston, 1983; Kushad and Yelenoski, 1987) was delayed by the application of coatings on apples in this study. This further confirms the suggestion that the lipid coatings used on apple and Nashi reduced the sensitivity of the fruit tissue to low temperature. Accumulation of putrescine was reported in some citrus fruits exposed to chilling stress (Yuen et al., 1995) and has been linked to the development of chilling injury in grapefruit, lemon and pepper (McDonald and Kushad, 1986; McDonald, 1989). The ability of putrescine to ameliorate various plant stress conditions such as acid (Young and Galston, 1983), osmotic (Wang and Faust, 1992) and mineral deficiency stress (Klein et al., 1979; Smith, 1984) and influence a wide spectrum of key metabolic activities (Galston, 1983) has led many researchers to believe that putrescine is a plant senescence inhibitor, and is produced in response to cold stress. 237 The accumulation and decline in the level of putrescine in this work coincided with that of a-farnesene in the uncoated apple. This was not the case in the coated fruits with the level of a-farnesene remaining fairly constant during storage at 0°C even though there were rapid changes in the putrescine production. If putrescine is associated with cold stress as suggested, it will be appropriate to assume that coating reduced the sensitivity of the fruit to chilling temperature, since the accumulation of putrescine did not occur until 15 weeks, 6 weeks after the control fruits. Since superficial scald did not develop before 9 weeks of storage at 0°C (as observed in pilot fruits during the course of the experiment) it will be reasonable to say that the development of superficial scald is dependent on the level of a-farnesene, which does not start to oxidise until after prolonged storage at low temperature. This therefore, explains why coated apples even though they had a decline in putrescine level, a trend similar to that of the uncoated fruit, still had no scald at the end of the storage. Apart from delaying the breakdown of the cell, the modified atmosphere generated by lipid coatings also reduced the production of a-farnesene within the fruit, subsequently controlling superficial scald. More work will have to be carried out to determine if the effect was as a result of low oxygen and/or high carbon dioxide levels within the fruit. Core and flesh browning of Nashi according to Beattie et al (1989) occur when fruits have been kept too long in cold storage. Core browning was reduced while flesh browning was prevented by coating Nashi with the oils and oil-based emulsions. Uncoated Nashi, although severely affected, had a carbon dioxide level less than 5% while the coated fruit 238 were above 5%. This result does not agree with the findings of Kader (1989), who reported that concentrations of carbon dioxide above 5% could induce core and flesh browning in pears during storage at 0°C. According to White et al (1990) the physiological life of the Nashi core is shorter than the rest of the flesh. At the end of the natural storage life, the core of the fruit turns brown and black, and then senesces while the flesh may remain unaffected. The core and flesh browning disorders in Nashi may be more of a manifestation of senescence than a result of high carbon dioxide levels. The modification of the internal atmosphere of coated Nashi delayed senescence, hence the effect on the development of the disorder. A similar hypothesis could be given for the prevention of brown heart in the coated fruits. The incidence of core flush was greater in the coated apples than the uncoated fruit which is contrary to the report of Neubeller (1971) in which waxing decreased core flush in McIntosh apples. This study, on the other hand, confirms the results of Wilkinson and Fidler (1973) and Smith and Stow (1984) who found that high levels of carbon dioxide caused core flush in coated stored apples. 239 6 CONCLUSION 6.1 Commercial potential of coatings Storage life extension of produce, such as apples and Nashi, by the use of lipid coatings, allow for improved export trading of these commercially important produce. Commercial scald inhibitors (DPA and ethoxyquin) have long provided excellent control of superficial scald in apples. The possible changes in antioxidant regulations in most countries, especially the US, emphasise the need to evaluate alternative scald control measures (Engle and D'Souza, 1989). This study has shown the possibility of using lipid coatings to control superficial scald of apples as well as some other physiological disorders in Nashi fruit. The application of oils directly onto the fruit or as an emulsion, in this case with sunflower oil, makes this coating technology applicable in both developed and developing countries. Only simple and relatively inexpensive- equipment is required. In the case of apple presently used waxing equipment can be used, replacing the wax with oil. In summary the newly developed lipid-based edible coating, due to their low costs and relatively simple application have a large number of functions. These include: 1. An extension in the storage life of a range of produce including lychee, banana, plum, nectarine, cucumber, avocado, apple, pear and Nashi fruit under ambient and low temperature (especially apple and Nashi) storage throughout the marketing chain. 2. Application of the coatings will help to maintain the quality of produce during transportation for home consumption in countries were adequate technology is not 240 available. 3. New markets for some products may be exploited where distance and cost of air or refrigerated transportation had previously made it uneconomical. 4. The benefits of reduction in post-harvest decay achieved in some fruit during CA storage may be extended to coated fruit during transportation and storage under ambient or refrigerated conditions. 5. Capital and energy cost can be reduced in refrigerated carriage and storage which, in some instances, may be dispensed with. 6.2 Commercial application of coating Mechanical waxing and drying equipment presently used for waxing apple would be used, replacing the wax with oil. The required amount of oil would be dispensed onto the fruit and passed over revolving brushes to spread the coating. The application of emulsions will require a dip tank with fruits being completely immersed in the emulsion using wooden crates or baskets. There is no evidence as to the effect of length of time for immersion of produce in the coating, but complete wetting of the fruit is essential. The fruit would be dried on a slat conveyor while passing through a hot air drier (if available) or just allowed to dry on a wire mesh or in wooden crates. Care should be taken that all fruits are properly dried before packing. Trash such as leaves and twigs that will possibly collect in the tank can easily be removed by lining the tank with a basket that can be raised after the operation for cleaning. 241 6.3 Potential problems The major problems associated with coating of fruits and vegetables are anaerobic respiration and failure of fruits to ripen properly. This will require more research in relation to modifying the concentration of the coating to the oxygen and carbon dioxide requirements of each produce. The coating materials could be washed off when there is need to ripen the fruit quickly or to reduce greasiness for market purposes. Commercial detergents such as the Tweens and Spans could be used. These are food grades. Further work is however required to determine the appropriate time of washing off the coating as this would be related to storage temperature and physiological maturity, as well as the concentration of detergents that will be just effective as not to cause injury to the produce. Repeated use of the emulsion could lead to contamination of the emulsion with decay organisms. The use of fungicides could therefore be considered for incorporation into the emulsion formulation. Since the lipid materials are prone to oxidative rancidity and hydrolysis more work is also needed on the long term storage and microbial stabilities of the emulsion. 6.4 Justification Although formal cost analysis is yet to be done on the production cost of the lipid-based coating developed, the easy availably of the ingredients and the quantities needed for the formulation justified the cost effectiveness. 242 As shown in Section 3.3 pg 81, the stock emulsion was prepared using lOg of sunflower oil (a 750ml bottle cost about $2.00), lg of Tragacanth gum (a 50g container cost about $30.00) and 88ml of water. The stock emulsion was further diluted as stated in section 3.4.2, pg 86 before use. A stock emulsion that will produce a 1L working (diluted) solution cost $1.01 while a stock solution of Semperfresh ( acommercial solution) that will be required to make a similar diluted working solution cost about $1.53. The coating application was by dipping, making it easy to apply, and the ingredients used for the formulation (oil and tragacanth gum) are all of food grade making the coated fruit save for consumption. 6.5 Recommendations The major problems observed during the preliminary studies i.e greasiness, anaerobiosis, uneven ripening and tissue breakdown, were overcomed in some produce using suitable types and concentrations of the coating materials. The types and concentrations of the coating materials fitted into the O2 and CO2 requirements of the produce. Based on the findings of this study the following is a list of i) produce that responded well to the lipid based coatings developed experimentally, ii) the appriopriate coating for each produce and the iii) possible storage life attainable based on a combination of factors such as colour changes, softening, weight loss and phsiological development. 243 Produce Treatments Potential storage life Apple Sunflower oil, marakot oil, 3.3% 20 weeks (0°C), 13 weeks (0°C) sunflower oil emulsion Banana Peanut, sunflower, marakot, or 18 days (20°C) coconut oils Carrot Coconut oil 35 days (20°C), 49 days (7°C) Capsicum Coconut oil, safflower oil 20 days (20°C) Cucumber Sunflower oil, marakot oil 29 days (20°C) Lychee Coconut oil 7-10 days (20°C) Nashi Sunflower oil, marakot oil, coconut 33 days (20°C), 11 months (0°C) oil, 3.3% sunflower oil emulsion. Nectarine Canola oil 15 days (20°C), 29 days (0°C) Plum Sunflower oil, marakot oil 9 days (20°C), 11 days (0°C) 244 7 BIBLIOGRAPHY Abeles, F.B. 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