BRUISING PROFILE OF FRESH ‘GOLDEN DELICIOUS’ APPLES

By

KAY M. MITSUHASHI GONZALEZ

Submitted is partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

WASHINGTON STATE UNIVERSITY Department of Horticulture and Landscape Architecture

MAY 2009

©Copyright by KAY M. MITSUHASHI GONZALEZ, 2009 All rights reserved

©Copyright by KAY M. MITSUHASHI GONZALEZ, 2009 All rights reserved

ii

To the faculty of Washington State University:

The members of the Committee appointed to examine the dissertation of KAY M.

MITSUHASHI GONZALEZ find it satisfactory and recommend that it be accepted.

___________________________

Carter Clary, Ph.D., Chair

___________________________

Marvin Pitts, Ph.D.

___________________________

John Fellman, Ph.D.

___________________________

Eric Curry, Ph.D.

iii

ACKNOWLEDGMENTS

I would like to thank the following people for their assistance with this project:

• Dr. Carter Clary, Dr. Eric Curry, Dr. John Fellman and Dr. Pitts – for his great support

and guidance throughout my Ph.D program at WSU.

• The Franceschi Microscopy & Imaging Dr. Valerie Lynch-Holm, Dr.Christine Davitt and

Dr. Michael Knoblauch – for their continuous help in the lab and long-lasting patience.

• CONACYT – for granting me a scholarship to be able to study at WSU.

• A mi mama, Gloria A. Gonzalez Aguila- por toda su ayuda, apoyo y amor. Mami gracias

por todo lo que me haz dado y ensenado a través de los anos.

• My sister, June Mitsuhashi- thank you for everything!

• Tyler Johnston – for being there for me all the time, patience and help.

• Johnston, Mclean, Hawkes and Ashburger family - for all their love and support.

• Edward Valdez- for his cheers and help during my research.

• Dr. Kohashi Shibata, M.S. Rodriguez Batista, Dr. Jorge Rodriguez Alcazar – for their

encouragement during the past years.

• Mr. Kyle Mathison, Stemilt growers, Andy Gale and Mike Robinson for the great

opportunity given to me.

• Mrs & Mr. Orahzda (Lucky Badger Orchards) - for all their support during the past years.

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BRUISING PROFILE OF FRESH ‘GOLDEN DELICIOUS’ APPLES

Abstract

by Kay M. Mitsuhashi Gonzalez Washington State University

May 2009

Chair: Carter Clary, John Fellman, Eric Curry, and Marvin Pitts

It is important to understand how apples bruise in order to prevent or reduce bruising. Tissue

from ‘Golden Delicious’ apples was analyzed to determine the bruising mechanism at different

maturity levels. Bruising was induced by an artificial finger attached to an Instron machine

applying an external load to fresh picked ‘Golden Delicious’ apples. To understand the bruising

mechanism involved, we used fluorescence microscopy with Calcofluor fluorescent dye to

identify cell walls and CDFA to identify cell membranes in the bruised and discolored tissue.

Together with SEM, different breakage mechanisms were observed in the bruised area. We

observed that 48 hours following damage, the bruised tissue was comprised of dead and live

cells, burst, crushed and intact cells. The more intercellular space there was in the tissue, the

more tissue damage occurred. Airspaces were the weakest points in the tissue structure, and

damage initiated in those points. As apples matured, there was an increase in damaged cells

surrounding larger intercellular spaces.

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TABLE OF CONTENTS

Page

ACKNOWLEDGMENT………………………………………………………………....………iii

ABSTRACT………………………………………………………………………………...……iv

LIST OF TABLES ………………………………………………………………………………vii

LIST OF FIGURES …….………………………………………………………………….…...viii

CHAPTER ONE

1. Introduction ................................................................................................................................ 1

2. Literature Review....................................................................................................................... 3

2.1. Fruit Anatomy and Bruise Potential .............................................................................. 3

2.2 Effect of Mechanical Injury/ Wounding and Types of Mechanical Stress on Bruising 10

2.2.1. Types of Mechanical Stress .......................................................................... 11

2.2.2. Mechanical Properties of Tissue ................................................................... 13

2.3. Physical Changes in the Cells ...................................................................................... 17

2.4. Chemistry of Bruised Tissue ........................................................................................ 21

2.4.1. Enzymatic Activity ....................................................................................... 23

2.5. Factors Affecting Bruising ........................................................................................... 27

2.5.1. Turgor Pressure ............................................................................................. 28

2.5.2. Firmness ........................................................................................................ 29

2.5.3. Maturity ........................................................................................................ 31

2.5.4. Harvest and Post-harvest factors ................................................................... 34

2.5.4.1. Evaluating Picker's Bruises on Bruise Color……………………35

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TABLE OF CONTENTS

2.5.5. Storage and Temperature .............................................................................. 37

2.6. Introduction to Microscopy.......................................................................................... 40

3. References ................................................................................................................................ 44

CHAPTER TWO: Bruising profile of fresh apples associated with tissue type and structure.

Abstract ……………………………………………………………………………………….....61

Introduction………………………………………………………………………….………...…62

Material and methods…………………..…………………………………………………...……67

Results.........................................................……………………………………………...………69

Discussion……………………………………………………………………………………..…70

Conclusions………………………………………………………………………………………72

Reference………………………………………………………………………………………...74

CHAPTER THREE: Harvesting by peel color to reduce bruising of ‘Golden Delicious’ apples.

Abstract ……………………………………………………………………………………….…84

Introduction………………………………………………………………………………........…85

Material and methods…………………………………………………………………………….87

Results………………………………………………………………………………..…………..90

Discussion…………………………………….………………………………………………….91

Conclusions………………………………………………………………………………………93

Reference………………………………………………………………………………………...94

SUMMARY ……………………………………………...…………………………………….100

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LIST OF TABLES

1.1. Frequency of disorders and diseases reported in USDA inspections of apple shipments.............................................................................................................................54

1.2. Other definitions of a bruise …...………………………………………………….….…..54

1.3. Average maturity indices for, some apple and pear cultivars at optimum maturity

stage…………………………………………………………………………………….…55

3.1. Fruit quality parameters for ‘Golden Delicious’ apples harvested at 3 peel color stages in 2007 and 2008……………………………………..……………………...…….96

3.2. Pearson correlation coefficients for fruit flesh firmness vs. imposed bruise volume

at 3 peel color maturity stages for’ Golden Delicious’ in 2007 and 2008…….……...…...98

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LIST OF FIGURES

1.1. Tissue elements of Malus (apple) fruit……….…………………………………………...55

1.2. Structure of a mature fruit……………………………………………………………..….56

1.3. Radial long sections from equator of the fruit cheek showing the skin at different

days from blossoming…………………………………………………….………………56

1.4. The timing of harvestable maturity within the normal developmental cycle of a

plant varies widely between individual products………………………...……………….57

1.5. Confocal scanning laser images and osmotically manipulated Granny Smith…………...58

1.6. CLSM images of apple cells dehydrated at 80°C at higher magnification…………….....59

1.7. Light micrographs of flesh from untreated and calcium treated

‘Golden Delicious’ apples………………..………………………...……………………..59

1.8. Schematic generic model linking the mechanical, structural and failure

properties of plant tissues…………………………………………………………………60

2.1. Discolored (bruised) area of compressed ‘Golden Delicious’ apple ……..………………77

2.2. FEI SEM images of control and bruised tissue……………………………..…………….78

2.3. Control and bruised tissue………………………………………………………….……..79

2.4. Confocal imaging of bruised and undamaged apple tissue………………………...……..80

2.5. A 45° crack from the applied force………………………………………..…….………..81

2.6. Crack parallel to the load…………………………………………………….……..……..82

2.7. Proposed bruising mechanism for ‘Golden Delicious’ apples…………………..………..82

2.8. Confocal image of cell wall and membrane damaged due to bruising……………...……83

2.9. Differece in intercellular spaces of fresh and stored apples ……………………….……..83

3.1. Bruise volume (imposed) at harvest as a function of year and peel color……………..….96

ix

3.2. Linear regression and Pearson correlation coefficient (r) for fruit flesh

firmness vs. imposed bruise volume for ‘Golden Delicious’ in 2007 and 2008……….....97

3.3. Grayscale light micrographs of ‘Golden Delicious’ apple peel

at progressive harvests in 2007 in size of intercellular spaces……………………………99

1

CHAPTER ONE

1. Introduction

Fruit and vegetable production is a multi-billion dollar global industry. The $2.5 billion U.S.

potato industry loses $300 million annually to bruising (Brook, 1996), most of which could be

prevented through proper conditioning and handling. Apple growers in New Zealand, South

Africa, and Sweeden lose a large amount of their production returns through bruising during

postharvest handling (Samin and Banks 1993; Viljoen et al 1996; Ericsson & Tahir, 1996 a, b).

It is estimated that $113 million are lost only in the US due to bruising (Varith, 2001).

Reduction in bruising in the fruit and vegetable industry could provide an annual payback of

billions of dollars (Baritelle and Hyde, 2001). The $1.2 billion U.S. apple industry, the $280

million U.S. pear industry, and other fruit and vegetable industries suffer considerable losses due

to bruising that could be reduced by proper conditioning and by improving the design and

operation of handling systems (USDA, 1999 in Baritelle and Hyde, 2001).

Bruising is the most important cause of rejection of apples at warehouses. The ability to

detect bruises depends on bruise visibility in the fruit, which in turn depends on bruise size and

color (Samin and Banks, 1993). Bruising may be caused by one or more types of loading

(compression, impact and vibration) during harvesting, transportation, sorting, packing and

storage (Table 1). The injured area is flat, soft, brown and has a high respiration rate. The bruised

tissue is also more susceptible to infection (Topping and Luton, 1986; Brusewitz et al 1991;

Ericsson and Tahir 1996 a,b). More than simply a visual flaw, bruising can also lead to bacterial

and fungal growth, lowering shelf-life. Studman (1997a) indicated that apple bruising can result

2

in product losses of up to 50% but normal loss levels are in the 10-20% range. Another problem

derived from bruising is water loss. Moisture loss of a bruised apple may be up to 400%

compared to that of a healthy apple (Wilson et al. 1995).

In the last decade, consumer demand for high quality apples has increased. Human

handling is the number one cause of external damage to fruit which begins at harvest. If the fruit

does not detach easily from the tree, the pickers who use two or three fingertips to pull the fruit

damage it. Growers prefer the use of the whole hand grip and a bending movement to

concentrate the force on the apple pedicel, not on the fruit flesh (Knee and Miller, 2002). Van

Zeebroeck et al. (2007) concluded that bruising is a major postharvest mechanical damage

problem for most fruit types.

Apples are subjected to various other loading conditions which can lead to bruising

beginning with their handling on the farm, down through the various stages of grading, storage,

distribution and processing, until eating. Between 20-50% of apples are bruised during handling

(Yuwana and Duprat, 1996). The apple industry suffers a lack of well-trained pickers. Growers

need their fruit picked and are forced to accept almost anyone to do so. One viable solution

would be to properly train pickers. Lack of pickers force industry to move towards mechanical

systems but extensive use of mechanical systems for harvesting and processing fruit and

vegetables, especially soft fruit, may increase the number of damaged or bruised fruit (Geoola et

al. 1994). Understanding bruising mechanisms in apples could help in the future to reduce this

kind of damage.

Bruising is not a result of a single factor that could be determined and applied across all

apple varieties. Bruising severity and susceptibility depends on apple variety, time of bruise,

harvest conditions, cause of the bruise, and many other factors. A number of researchers have

3

studied the factors affecting bruising (Klein, 1987; Saltveit, 1984; Schoorl and Holt, 1980;

Topping and Luton, 1986), but very few have followed what happens over time with bruises

under storage conditions (Ingle and Hyde, 1968; Toivonen et al. 2007). Bruise damage could be

reduced by decreasing bruise susceptibility of the fruit by modifying growth and handling factors

(Hyde and Ingle 1968; Diener et al 1979; Holt and Schoorl 1977c; Klein 1987; Brusewitz and

Bartsch 1989; Hung and Prussia 1989; Johnson and Dover 1990; Banks and Joseph 1991).

In 2006, Stemilt Growers Inc., Wenatchee, WA, had bruising up to 60%

per bin (capacity approximately 1000 lbs). Since 2005, Stemilt has tried to

control its bruising by implementing programs to reduce bruise damage. Downgrading apples

due to bruising affects growers as the price paid is less for the damaged apples. The main

purpose of this research is to understand the mechanisms of bruising in the different maturity

stages based on skin color in ‘Golden Delicious’ apples. ‘Golden Delicious’ apples are usually

single picked, they can be picked when their skin color is green or yellow once they reach their

maturity index. Understanding bruising at different skin color stages could help reduce bruising

when picking based on skin color.

2. Literature Review

2.1. Fruit Anatomy and Bruise Potential

Apples (pomes) are fleshy fruit derived from an inferior ovary (MacArthur and Wetmore,

1939; 1941; Smith, 1940, 1950). An apple is considered to be the extracarpellary part of the

pome fruit; the flesh of the fruit comprises the floral tube and capillary tissue. The floral tube

4

region consists of fleshy parenchyma cells with a ring of vascular bundles. There are five

petallary and five sepallary bundles, alternate with one another. Branches from these bundles

permeate the parenchyma cells as an anastomosing system. The sub epidermal parenchyma of

the floral tube region comprises several layers of tangentially elongated cells with thick walls.

There are no intercellular spaces until the later developmental stages. The ground parenchyma is

located deeper in the tissue and has significant intercellular spaces. The still deeper-lying ground

parenchyma cells are roughly elliptical in outline with a radial orientation. This part of the fruit

undergoes intensive growth during development, first by cell division and cell enlargement, and

later by cell enlargement only (see Figure 1).

The ovary wall is differentiated into the fleshy parenchymatic exocarp and the

cartilaginous endocarp lining the locules (MacDaniels, 1940 cited by Esau, 1965). The exocarp is

comprised of parenchyma cells. The cartilaginous endocarp is sclereids with such thick walls that

the cell lumina are almost occluded. In the region of the median carpellary bundles,

sclerenchymatic cells are absent so that the hard endocarp of each carpel forms two disconnected

sheets of tissue, one on either side of the locule. The cartilaginous endocarp is the first tissue of

the apple to attain maximum development (see Figure 2). The fleshy exocarp is next, followed

by the extracarpellary tissue. The latter continues to grow up to complete ripening of the fruit

(Esau, 1965). Depending on the type of fleshy fruit, the external ovary wall differentiates into

parenchymatic tissue and the cells retain their protoplasts in the mature fruit. Immature fleshy

fruit walls have a firm texture and become softer with ripening (Winkler, 1939 cited by Esau,

1965). Chemical changes in the cell and structure of the walls cause softening in the fruit (Reeve,

1959), and the cells detach from each other.

5

The skin is an outer protective tissue of the fruit, comprised of cuticle, epidermis and

hypodermis. At harvest, cuticle thickness varies among cultivars In ‘Golden Delicious’ the

cuticle thickness is around 10µm (Meyer, 1944). Epidermal cells are larger radially than

tangentially at bloom, but later they become tangentially elongated (see Figure 3). In ‘Golden

Delicious’ apples, the epidermis identity is lost at maturity. At bloom the hypodermis cells are

very similar to the cortex, but smaller (Tukey and Young, 1942). Later, the cell wall thickens and

elongates tangentially, but the difference between hypodermis and cortex can still be difficult to

identify (Skene, 1962). Cell separation occurs during growth of apples. At maturity 25% of the

fruit volume is air space between cells (Khan and Vincent, 1990). This increase in air space may

account for the decline of firmness due to detachment of the cells during growth and is difficult

to separate from ripening- related changes in fruit texture (Knee, 1993). As fruit ages, the volume

fraction of airspaces increases, cells grow and are pushed apart (Tetley, 1931). During ripening

and senescence, there is a continuous decrease in cell adhesion which leads to the detachment of

cells and an increase of intercellular spaces (Knee and Bartley, 1981). This gives apples a mealy

texture. The size of the intercellular space is a tool to define the age of the fruit. Firmness is not a

reliable indicator for best harvest dates because this measurement is affected by the mechanical

strength (Hertog et al. 2003) and the physical anatomy of the tissue, such as cell size, cell shape

and tissue arrangement and also depends on the apple variety (Toivonen and Brummell, 2008).

The fruit epidermis is comprised of small tightly packed cells with a thick outer cuticle.

The surface of many fruits is smooth and waxy. This could decrease the frictional contact

between fruits and other surfaces. Beneath the epidermis, the hypodermis may contain up to six

layers of collenchyma cells, which contribute to the strength of the epidermis (apple peel/skin).

The strength of the apple skin provides protection against puncture, but not against impact

6

because the inelastic outer cell layers transmit the force to the underlying parenchyma cells.

Some fruits have a much thicker skin, providing the underlying tissues more protection against

different types of mechanical stress. In citrus fruit, the loose cell packing in the albedo layer of

the peel is ideal for absorbing impact loads (Knee and Miller, 2002). Unlike citrus fruit, the

epidermis of the apple fruit consists of radially elongated cells during its younger stage, while at

maturity the tangential diameter surpasses the radial. Thus, as the apple matures, the cuticle on

the epidermis increases in thickness (Tetley, 1930, 1931). Granules of wax accumulate on the

cuticle (Mazliak and Chaperot, 1959 cited by Esau, 1965). The flesh of an apple is mainly

parenchyma cells (Roth, 1977 in Knee and Miller, 2002). These are approximately 100 µm in

diameter, with cell walls about 1 µm thick and unlignified. These thin walls are a fragile part of

the plant cell. Turgor pressure in fruit tissue which is the osmotic potential of the organic acids

and sugars in the vacuole that occupies most of the cell volume provides the rigidity of fruit

tissues. The parenchyma cells in many fruits are less isodiametric (several equal diameters).

Radial arrangements of the cells themselves may be obvious, as in kiwi fruit, peaches and

strawberries; or only apparent on microscopic examination, as in apples. The weakness of fruit

parenchyma tissue is due to loosely packed cells with large intercellular spaces between cells

(Knee and Miller, 2002). Larger intercellular spaces can be points of higher stress in the tissue,

were damage can occur easily.

Fruit parenchyma cells are among the weakest plant cells because the cell walls contain a

low proportion of cellulose microfibrils arranged in a gel matrix of pectic polysaccharides.

Harker and Hallet (1992, 1994) reported that apple and kiwi fruit cells burst at an internal

pressure of 0.5 to 1 MPa. Even though the failure stress of the cell walls is high (1MPa to

0.5MPa), fruit tissue can be injured at only 0.1 MPa of stress pressure. Following such damage,

7

the change of shape and volume result in cell wall voids which allow the release of water,

depending on the permeability of the plasma membrane and cell wall to water ratio. Wu and

Pitts (1999) concluded that the extrusion under semi-static loading would require a strain rate of

0.0001 s-1. Wu further states that rapid loading, which occurs during impact, would not provide

enough time for extrusion, therefore cell breakage would be common.

Although parenchyma cells are among the weakest plant cells, they are specialized to

withstand certain specific stresses (Brett and Waldron, 1990). In stems, vertically oriented walls

show a tendency to transverse orientation of microfibrils, which allows the cell to bend without

breaking. Other features that increase mechanical stability include the presence of structural

elements like vascular strands and fiber or sclereids. These are more common in drupes (peach,

plum and mango) and pomes (apple and pear) than in berries (grape, tomato and banana) (Knee

and Miller, 2002).

Cells may be broken due to stress in a concentrated area of the cell wall. Under under

gross tissue loadind or compression and shear, stress will affect the contact area of the cells.

Under shear, the cell breaks if the adhesion between cells is stronger than the cell walls. Cell

adhesion depends on the properties of the middle lamella, which is weaker than the cell wall

because it contains pectin only and no cellulose. Tensile testing in immature fruit tissue have

shown break stresses of 0.34 MPa for apples (Harker and Hallett, 1992). Failure in this instance

was due to cell breakage and the presence of airspaces. The middle lamella showed no breakage

due to the cell-to-cell contact in the tissue (Knee and Miller, 2002). Cell breakage or detachment

of the middle lamella will depend on the tissue properties.

Fruit tissue shows viscoelastic properties, and permanent deformation (injury) depends

on the rate at which the force is applied. When slow loadings are applied plastic deformation

8

occurs as long as the elastic limit is not reached. Once the limit is surpassed failure occurs.

(Knee and Miller, 2002). Therefore, cells can absorb energy depending on the limits of their

elasticity. Because most of the elasticity of a cell is due to the turgor pressure of the cell content,

Steudle and Wieneke (1985) demonstrated that high turgor pressure makes cells brittle, thus

impact energy must be absorbed by other means than elastic deformation. If turgor pressure in

cells is lowered, they can deform elastically and return to their original shape after the stress is

released. Reaching the elastic limit results in plastic deformation. This happens at different

levels: 1) microfibrils may slip in the matrix of the cell wall; 2) cells may slip against one

another along the line of the middle lamella; 3) cells may be broken damaging tissue.

Ripening involves the loss of cellular adhesion and weakening of the primary cell wall

with the help of polysaccharide degrading enzymes. Immature fruit contains starch reserves that

are hydrolyzed to sugars during ripening, thereby increasing the osmotic potential of the cell

content, which leads to an increased turgor pressure if the external water potential is high. Fruit

cells are unusually prone to rupture in solutions of low osmotic strength because of their weak

cell walls (Simon, 1977). In healthy (intact) fruit, there is an equilibrium of intracellular solutes

so that turgor forces do not disrupt the cells as wall pressure declines due to ripening. As a

consequence of cell wall and turgor changes, the internal tissues become less elastic and more

plastic with ripening. Tissue becomes weaker with ripening. Therefore, it is common to have

more injury with a given external mechanical stress as the fruit ripens. The mode of failure under

mechanical stress may differ so that cells separate, and less cell breakage occurs in the ripe fruit

than in the unripe fruit (Harker and Hallett, 1992, 1994; Harker and Sutherland, 1993). The cell

membrane is a selective barrier for the movement of compounds within and between cells. This

membrane controls biochemical and physiological process in the cell. During senescence, there

9

is a loss of membrane integrity and control of what comes in and out of the cell is lost

(Thompson, 1988). A marked change in fluids in the membrane occurs with senescence. Fluidity

decreases and the membranes become more rigid. This alters the enzyme activity associated with

the membrane and its receptors.

Cells separate in ripe fruit at strains of 0.08 MPa for apple and 0.01 MPa for the other

fruits (Harker and Hallett, 1992, 1994; Harker and Sutherland, 1993). Cells in ripe fruit under

mechanical stress may slide against each other to relax the stress without breaking the cells.

Unlike previous research, Harker and Hallett (1992) and Steudle and Wieneke (1985) concluded

that the cell walls of apples actually become stronger during ripening (Knee and Miller, 2002).

This contradicts the ripening process of cell decompartmentalization (Kays, 1997) which makes

cells more susceptible to damage, more brittle. This finding could help identify what is the best

time to harvest based on greener or ripper apples, making fruit less prone to damage when

harvested.

Edible plants have three primary tissue components that determine the fruit’s mechanical

behavior; parenchyma cells; intercellular bonding between cells; and intercellular space between

cells . Turgor pressure and cell wall strength control the mechanical properties of parenchyma

cells. Turgor pressure within the cell is regulated by the bidirectional movement of water through

the plasma membrane. Water movement allows the intercellular and internal water potentials to

equilibrate. The cell wall restrains the outward stretch of the protoplasm which is structurally the

most rigid component of the cell. Parenchyma cells only have a very thin primary wall and no

secondary cell wall in apples. Stiffness of the primary cell wall is conferred by the cellulose

microfibrils; the wall is very thin and will bend easily (Kays, 1997).

10

The cell walls of ripening fruit also cause color changes. Immature fruits have

chloroplasts in the outer cells and are therefore green. Chlorophyll disappearing and carotenoid

developing pigments induce color changes to yellow, orange or red. Ripening fruit may form

anthocyanins causing the tissue to turn red, purple or blue. These pigments are distributed in the

fruit wall, like in some cherries, or only on the peripheral parts of the fruit wall, as in plums or

Concord grapes. The outer epidermis accumulates tannins (Esau, 1965). In apples, chlorophyll is

present in young immature fruit in epidermal cells and to a less extend in the cortical cells of

pomes (Ryugo, 1988). The reason some apples remain green is due to the high chlorophyll

content, but its concentration decreases with maturity. In fruit that turns yellow or red as they

mature, chlorophyll is degraded and disappears so other pigments become visible. In red skin

apples, the skin will turn deep red due to its anthocyanin content. Epidermal cells of light

skinned apples will develop a red blush only when exposed to direct sunlight. Anthocyanin

synthesis is increased in apples when day temperatures are relatively warm and night

temperatures drop to 7°C to 10°C. Cool night temperatures reduce respiratory loss of

carbohydrates in the epidermis and light favors pigment formation.

2.2 Effect of Mechanical Injury/ Wounding and Types of Mechanical Stress on Bruising

Mechanical stress is physical injury to fruit. This is the most important type of quality

loss during harvest and postharvest handling. Damage to fruit tissue causes an increase in

ethylene production and respiration, providing a site for decay organisms to enter the product

(Ceponis et al.1962). Mechanical injury in apples is the primary cause of postharvest loss. Such

11

loss is due, in part to, loss in weight due to an increased respiration and water loss

(physiological), and by infection caused by organisms that enter the product (pathological).

In order to reduce the damage caused to apples by mechanical injury the energy applied

could be reduced by training picking and packing personnel and /or padding hard surfaces, but

probabilities of cell failure such as breakage or bruising could still increase as fruit ripens. When

the cell walls start to fail due to cell decompartmentalization, the impact energy stored in the

tissue could be transferred to the other neighboring cells. The probability of failure is more

common at a lower energy applied than at impact (Knee and Miller, 2002). Due to fruit tissue’s

viscoelastic properties, the magnitude of the deformation (injury) depends on the rate at which

the force is applied. Slow loading permits plastic deformation when the elastic limit is reached.

2.2.1. Types of Mechanical Stress

There are three major means by which the mechanical damage can occur. They include

compression, impact and vibration forces (or friction). Compression forces during harvest could

be applied by the picker’s hands and fingers, or with their legs, when leaning against the picking

bag and ladder, and when the fruit is dumped into the bottom of the bin. External damage can

also be caused by other apples due to the weight of the picking bucket, deep bins or when

forklifts haul the fruit. At the warehouse, compression, impact and vibration can occur while

packing the fruit in boxes, as well as when washing and waxing the fruit. Compression is

common during transportation and storage. Damage includes splitting of the product, for

example in tomatoes whose diameter is expanded to the failure point; in potatoes, internal shear

has been observed (Sherif, 1976) resulting in permanent deformation of the damaged area.

12

Finally, once the fruit reaches market, some buyers like to squeeze or pick through the fruit in

order to check for firmness and freshness, causing a depression of tissue in the apple (Brown et

al. 1993).

Friction is the result of fruit moving against each other, resulting in surface abrasion. This

damage is common in grading belts and during transportation. Friction involves transmitted

vibration when hauling and moving the product. Friction damage is found on the epidermis and

underlying layer cells. The surface tissue darkens due to the enzymatic oxidation of the injured

cells contents. This site is considered an entry port for fungi and other unwanted microorganisms

(Kays, 1997).

In order to understand mechanical damage and stress in fruit and vegetables, it is

important to understand changes at the cellular level. When an external load is applied to a fruit

or vegetable, the cells begin to change shape at the site of the applied force, reducing the

diameter of the cell in the force direction. The cell contents do not compress when the force is

applied, or compress very little, yet the change in shape alters the surface to volume ratio of the

cell, increasing its turgor pressure. To equilibrate the internal and external water potential, some

water moves to the outside of the cell to balance the increased turgor pressure. If the external

load applied is low and of a short duration, most cells will recover their original shape, making

the deformation largely elastic. Compression of longer duration will cause a greater net efflux of

water and a lower recovery in the cells. If the stress is too large, the cells will rupture because

the strength of the walls is exceeded (Kays, 1997).

13

2.2.2. Mechanical Properties of Tissue

Intercellular bonds in cells are important to the fruit’s mechanical properties. Cells are

attached by a pectin layer known as the middle lamella. As previously mentioned, the middle

lamella is flexible and cells are allowed to change position when a slow compression load is

applied. When the shear stress in the middle lamella is exceeded, the cells separate without

rupturing. In cell detachment, the cells begin to separate at a 45° angle of the direction of the

force applied which results in a visible gap (break) in the tissue like in potato tissue (Kays,

1997).

Intercellular space or airspaces provide strength to the tissue. Between the cells, there are

airspaces and intercellular fluid. If the airspaces are large, for example in peaches, it gives the

cells space to reorient when a compression force is applied. With this reorientation, the tissue

volume changes. In dense products like potatoes, the airspaces are small, and there are few

rearrangements of the tissue and little compression of the tissue (Kays, 1997). Apple tissue, in

general, consist of uniform cell size and airspaces (Alvarez, 2000).

There are four modes of tissue failure when an external load is applied: cleavage, slip,

bruising, and buckling. Cleavage occurs when failure is at the maximum shear stress, resulting

in two separate and intact pieces. When the compression force results in failure along the tissue

at a 45° angle to the direction of the load, the two pieces slip or slide along the airspace, this

failure is called slip. This is common in apples and potatoes. Bruising is the most common

failure mode in fruits and vegetables and it occurs when the cells rupture and expose their

internal contents to the air and enzymatic oxidation occurs, causing discoloration and browning

of the tissue (Diehl, 1980a,b).

14

Bruising is defined in the literature in numerous ways (Table 2). According to Mohsenin

(1986) a bruise is the result of a physical and or chemical change in fruit, which affects flavor,

color and texture. This could be a result of external forces that occur during harvest,

transportation, handling and packing. Detecting a bruise in apples is difficult because the skin

hides the underlying damage especially in red varieties.

Bruising is evaluated by direct visual characteristics, like discontinuities, fractures, or

changes in shape and size of the tissue (Chen et al, 1986; Garcia, 1988; Garcia et al. 1988; Holt

and Schoorl, 1977 a,b; Hyde and Ingle 1968; Rodriguez 1988; Topping and Luton, 1986).

Impact load creates small bruises in fruit while compression forms large ones. In the bruised

area discontinuities become visible below the skin (a few millimeters from the surface) where

maximum stress applied (Chen et al, 1986; Garcia,1988; Rodriguez, 1988). Beneath the

epidermis the stress and maximum deformation are the greatest. This area equals the radius of

the indenter used to damage the fruit (Horsfield et al. 1972; Miles and Rehkugler, 1971).

Holt and Schoorl (1977b) defined bruising as the bursting of cells under compression.

When impact under dynamic conditions occurs, the cells straighten out reducing in diameter and

snap, like a slack rope does when it is suddenly loaded. More energy is diffused in breaking

microfibrils increasing and bruising. Rodriguez et al. (1990) found that impacted tissue showed

a large number of injured cells in the central region, 9 or 10 mm under the skin, but no cell wall

rupture. The middle lamella was observed to split in epidermal cells beneath the parenchyma

cells. Inside the bruised area intense, vesiculation was visible in the middle lamella region

between cell walls of adjacent cells and in the central vacuole. Membrane breakage was more

visible in parenchyma cells under the epidermis than in the epidermis itself. Rodriguez et al.

(1990) did not find any cell wall rupture of parenchyma cells. The tissue showed deformation

15

when compressed that was flatter in the direction of the load applied (compression).

Deformations were more common in the central parenchyma, approximately 8 to 9 mm

underneath the epidermis. Cell degradation and tonoplast breakage were observed, and in other

wall compounds vesiculation occurred. In the central area of the bruise, deposits of unknown

material were found. Some of the cells underneath the epidermis showed cracks in the middle

lamella region between adjacent cell walls, but there was no cell wall rupture observed.

The difference in structural response in apple tissue under compact and impact loads was

found to be significant (Rodriguez et al., 1990). The damaged cells developed from severe

vesiculation in the vacuole and region between cell walls of adjacent parenchyma cells showed

cracking of cell walls of adjacent parenchyma cells. The authors hypothesize that vacuolar

vesiculation under impact was due to the momentum of physical forces observed during impact

load, and that the cytoplasm might pull back the vacuole forming vesicles. Cell wall vesiculation

could not be explained. Due to the short time lapse (2 hours) between tissue fixation and the

external load applied it is not probable that the synthesis of cell wall enzymes occurs causing

degradation and vesicle formation. Rodriguez et al. (1990) observed that compressed cells show

a deformation in the direction of the external load, which confirms the model postulated by Chen

et al. (1986); Dal Fabro et al. (1980); Holt and Schoorl (1977b); Nilsson et al (1958); Pitt (1982);

Segerlind and Dal Fabro (1978) that cells under compression or slow loads experience a change

in shape, getting smaller in the direction of the applied load, while filling the intercellular spaces.

The cell walls behave elastically, returning part of the energy applied, and absorbing some as

they change shape, getting flatter, and resulting in permanent deformation. Compression forces

play a role in moving degradation materials from the cell wall from higher pressure areas to

lower pressure areas. It was observed (Rodriguez et al., 1990) the stress produced and the

16

development in the tissue was different under impact and compression loads. Under

compression, cells show deformation in the direction of the applied force, under slow load

changes in shape occur. The cells begin to fill the intercellular space staying permanently flat.

During impact, cells split by the middle lamella and vesiculation in the vacuole and middle

lamella region occur. Further research to clarify what causes vesiculation under load is needed

as well as biochemical studies to identify enzymes and substrates that would be needed to

determine if the browning reaction could be produced in different ways under different loads.

The majority of the bruises as a result of impact under a small drop height formed lines of

cell ruptures at certain depths. Cell rupture is present in the weakest cells, which are not those

necessarily closest to the skin (Yuwana and Duprat, 1997). The ruptures are not always arranged

horizontally, while the cells near the skin remain intact. This demonstrates the heterogeneity of

fruit tissue responses to impact (Duprat et al. 1991 cited by Yuwana and Duprat 1996). The

damage is due to the fact that cortex cells are parenchyma cells which are weaker and have a

thinner cell wall compared to collenchymas cells. Cortex cells are found in the first six layers of

apple fruit and have thicker, stronger cell walls (Esau, 1965).

Although some researchers have proposed that bruising in fruit like peaches occur through

shear below the skin of the fruit (Horsfield et al.,1972) others argue that apple tissue is unlikely

to fail in this way under compression because radial airspaces in the fruit’s parenchyma allow

stress to be relieved by lateral movement of cells (Khan and Vincent,1990). Apples contain 15-

20% airspaces whereas peaches and nectarines have less than 10% airspace (Hatfield and Knee,

1988; Harker and Hallett, 1992; Harker and Sutherland, 1993). Shear failure is more common in

peaches due to the lateral movement of cells being restricted by their lower proportion of

airspace in the radial arrangement of vascular strands. Compression failure could be a process in

17

which energy is absorbed by a mass of cells breaking simultaneously. Shear is a more gradual

process in which the cells absorb energy by breaking over time. With shear, there may be a

higher ‘crack stopping’ and distribution of damage without apparent bruising (Vincent, 1990). It

may be relevant that apples tend to show sudden failure under compression, whereas peaches fail

more gradually as stress increases (Fridley and Adrian, 1966).

Wounded tissue can rapidly deteriorate and die or it can heal at the damaged site. How

the tissue acts depends on the fruit characteristics (Kays, 1997). Wounded tissue can heal by

replacing the damaged tissue as in the apical meristems, by induction of secondary meristems

(wound periderm formation in tubers), or by changes in the physical and chemical composition

of neighboring tissue to the damaged cells. Younger meristematic tissue heals more easily than

older. When apples are still attached to the tree, they will heal wounds until mid-July, after

which wound healing ability diminishes. Stored apples do not have any healing abilities at all.

Desiccation in damaged tissue occurs as a response to wounding. Parenchyma cells close to

the wound suberize, and some lignify below the desiccated cells. Cell division is stimulated

below the damaged area to form cambium (phellogen), and continued cell division forms cork

(phellen). In some tissues there is no phellogen formation, only suberization of the neighboring

cells.

2.3. Physical Changes in the Cells

The damage susceptibility of fruit is determined by its mechanical properties, which

depend on many microscopic histological and cellular features such as type of tissue, geometric

18

properties of the cell, presence of an adhesive middle lamella between cells, mechanics of the

cell wall, cellular water potential and presence of intercellular spaces, among others. (Van

Zeebroeck et al. 2007). Oey et al. (2006) placed a microtensile stage under a stereomicroscope

so they would be able to observe the deformation of individual cells under compression.

Variations were found for deformations of individual cells under compressive or tensile loading,

even within the same tissue. A clear influence of turgor on some parameters could be observed.

These parameters were length and width. In tensile tests, cells became significantly longer and

thinner when the tissue had low turgor. Cells in soft tissue had larger deformations than cells in

turgid tissue at similar stress levels. In compression tests, the same effect was observed, but in

the opposite direction: cells become wider and shorter, and the influence of turgor was not as

specific as in tensile tests. Shortening of cells in the tissue was not significantly different among

various turgor levels. In relation to cell width, deformation in turgid samples was smaller, but no

difference was found between cells with normal and low turgor. Area and perimeter did not

change when cells experienced deformation in length and width. These parameters became

smaller in compression and larger in tensile tests. These authors found cell liquid was

compressible to a limited extent. A change in area has to be associated with an increase or

decrease of cell depth. A clear influence of turgor on area and perimeter was not found in the

tests. Roundness of cells was lost in both kinds of testing, but increased when turgor is reduced.

This effect is not found for fresh apples under compression (Oey et al. 2006) For tissue of

stored apples, the influence of turgor on the deformation of cell structure parameters is the same

as in fresh tissue, but under compression flaccid cells experience larger deformations compared

to cells with turgor. Turgor manipulation effects to a greater extent the deformation of cells in

stored tissue, where the integrity of cells is reduced. In tensile tests, turgor has a larger effect on

19

cell deformation in fresh tissue. Deformations in stored tissue were smaller for both tests.

Cellular deformations in length were closely related to strain measurements in both tests, but the

process of cell deformation was different in tensile tests. The load is evenly distributed over all

the cells in the tissue sample. In compression tests, the whole tissue gets considerably more

compressed than the individual cells. It seems that the cells closer to the grips of a worker’s

fingers (surface) are compressed before the cells in the middle of the sample. In compressive

load the load is not evenly distributed within the sample, but goes from the outer cells to the

middle of the sample. Collapse of the tissue goes from the exterior cells, close to the grips, to the

interior.

In contrast to compression tests, the stiffness decreases also at higher strains when turgor

is lowered. For failure stress, a slight increase in turgor was found, but differences were not

significant. This finding contrasts with results on pears, where strength of the fruit was related to

its firmness (De Belie et al. 2000). The increase of strength was attributed to stress-hardening

through turgor, and resulted in rupturing of individual cells. Tensile strength of soft pears has

been shown to be independent of turgor, and has been related to cell-to-cell debonding at the

middle lamella.

Oey et al. (2006) found that the stress-strain curve of fresh apples shows a great decrease

in force at failure. This is a characteristic of cell rupture. The connections between the cells

through the middle lamella are strong enough to tear cells apart. In fresh apples, failure is

independent of turgor, and only depends on the strength of the cell wall. The cell wall is not

altered by controlling the turgor by water uptake or release, nor does it experience stress-

hardening, or pre-stressing leading to increased brittleness. If alterations of the cell wall occur

due to turgor manipulation, this cannot be detected using tensile tests. In stored apples, adhesion

20

between the cells is reduced due to pectin solubilization in the middle lamella. The subsequent

loss of structural integrity and the loss of cell fluids by exudation cause lower stiffness in stored

apples (Varela et al. 2007). This explains why stress and strain values found for stored apples

were lower as compared to fresh apples. The influence of turgor was the same, but strain

increases and stiffness decreases when turgor is reduced, and no significant effect was found on

failure stress. At failure, force declines slowly due to cell-to-cell debonding. Using scanning

electron microscopy Tu et al. (2000) observed a clear separation of apple cells when the tissue

had a tensile strength of 0.06-0.08 MPa. This corresponds to the values that Varela et al. (2007)

found for tensile strength of stored apples. For stored apples, tissue failure in tensile tests is due

to cell-to-cell debonding and the point at which failure occurs is determined by the strength of

the middle lamella, not by turgor. According to Oey (2006), Varela et al. (2007) and Tu et al.

(2000), when a force is applied to stored and fresh apples, there is a different failure mechanism.

Stored apples cells separate at the middle lamella, but in fresh apples it is what mechanism that

depends on cell wall strength, not the strength of the middle lamella.

Lin and Pitt (1986) described failure mechanisms in apple, the influence of loading rate,

and water potential on the mode of failure where there are three possible types of failure:

plasmolysis (cell rupture due to the absorption of water), cell debonding (breaking apart of the

cells such that the cells change position with regard to one another perhaps combined with

rupturing cells), and cell rupture (based on a model).

21

2.4. Chemistry of Bruised Tissue

A bruise develops after an external load is applied to a fruit (Coombe, 1976). Bruising is

evaluated by the amount of softened tissue and discoloration present in the tissue. The cell walls

are degreaded by polygalacturonases, pectinmethylesterases, and cellulases. These compounds

responsible for causing cellular softening (Arie and Noemi, 1979 cited by Rodriguez et al. 1990).

Discoloration of the tissue is due to an oxidative reaction where polyphenoxidases oxidize

phenolic compounds to quinines that are unstable and polymerize as melanic compounds with a

high molecular weight (Vamos-Vigyazo, 1981). Polyphenols are located mainly in cell

vacuoles, and polyphenoxidases in the chloroplasts (Vamos-Vigyazo, 1981). Phenolic

compounds are possible substrates of catechol oxidase (phenolase, polyphenoloxidase) which are

located in the plastids of healthy/intact cells. When cells break, tonoplasts rupture, and substrates

and enzymes in the cell mix. If molecular oxygen is available, catecholase attacks O-diphenol

groups forming quinines and water. Quinines polymerize into dark colored compounds. The

optimum pH of catechol oxidase is 5 to 6. Reactions may be delayed by organic acids that are in

the vacuoles of many fruits. The organic acids lower the ph (Mayer and Harel, 1981). Ascorbate

can reduce quinine groups into hydroxyls.

In apples, bruised tissue darkens in minutes. The intensity reaches its maximum point 10

to 20 hours (Samin and Banks, 1993), in contrast to the three to four days reported by Duncan

(1973). The bruise development can be accelerated by elevating the temperature. Later, the

visibility of the bruise diminishes (Samin and Banks, 1993). According to Samin and Banks

(1993), the dry weight of the bruised tissue decreases by 95% after 10 hours. Apparently, solutes

22

are absorbed into the adjacent healthy tissue (Samin and Banks, 1993). The bruised area

reduction is related to a gradual water loss due to evaporation or absorption in the tissue.

Biochemically, bruise development is due to phenolic content and oxidative enzymes,

mainly polyphenol oxidase (PPO), but also peroxidases (Duncan, 1973). In immature fruit, high

levels of phenolic compounds are present. During maturation and ripening these compounds

decrease (Mayer and Harel, 1981; Ozawa et al. 1987). Weurman and Swain (1955) found that

the potential browning caused by changes in phenol concentration of apples is higher than the

actual browning. The concentration of phenols in the tissue is the determining factor in

enzymatic browning (Guadagni et al. 1948; Kertesz, 1933; James and Crang, 1948 in Weurman

and Swain,1955). Weurman and Swain (1955) suggested that potential browning should be

considered a good indicator of enzyme activity when the natural activators and inhibitors are

present. The values for potential browning correlate with, but at a much higher level than, those

for the actual browning. In situ enzymatic activity can be considered an even more important

factor contributing to the intensity of browning than the concentration of phenol substrates. The

concentration of ascorbic acid in the cell regulates the activity of the enzyme in a manner similar

to that found by Baruah and Swain (1953) in vitro. It would be expected that the actual

browning, and the potential browning, as well as the enzyme activity, would be inversely

proportional to the concentration of the vitamins. If it is assumed that the intensity of the

browning at any stage is dependent also on the amount of phenols, the ratio of phenols/ascorbic

acid would be expected to be proportional to the browning. Although the concentration of

ascorbic acid may have an effect on the actual and potential browning, it does not influence the

enzyme activity as Baruah and Swain (1953) showed.

23

The oxidative reaction that develops in the intercellular spaces after cell rupture is due to

mechanical action (Diehl et al., 1979; Pitt, 1982). Faust and Shear (1968) and Shoei (1984)

affirmed that discoloration (browning) occurs inside the cell, in the cytoplasm. The metabolic

activity of bruised tissue is limited to early stages and the darkening reaction seems to occur with

cell death, in contrast to wounded tissue that continues induction of defense proteins and is not

followed by cell death. In none of the cases is there induction of PPO, but in impacted cells,

there is a subcellular relocation of PPO related to melanic deposits as the bruise develops. When

impact damage occurs, cell death is apparent. The magnitude of the discolored area is influenced

by the impact severity (McGarry et al. 1996). McGarry et al (1996) found that cell death initiates

approximately 3 hours after the application of an external load. This onset is simultaneous with

decompartmetalisation. The appearance of lipid peroxides occurs in 1 hour, as does the start of

melanin production. Melanin production causes cell death by tanning cellular constituents and

preventing their function. Long term effects of bruising include cell death, dehydration and large

spaces, compromising the fruit’s natural barriers against possible infection. Wound healing

heightens metabolic activity and repair of the damaged tissue where there is no extensive cell

death. Luluai et al. (1996) confirmed that with bruising there is an absence of wound healing,

leading to cell death.

2.4.1. Enzymatic Activity

Enzymatic browning is a very important color reaction, which affects fruit, vegetables and

seafood. It is estimated that more than a 50 percent loss in fruit occurs due to enzymatic

browning (Whitaker and Lee, 1995). Browning in tissue has an effect on consumers when

24

purchasing, and a negative effect in flavor and nutritional value. Enzymatic browning reaction is

catalyzed by the enzyme polyphenol oxidase (1,2 benzenediol; oxygen oxidoreductase), also

known as phenoloxidase, phenolase, monophenol oxidase, diphenol oxidase, diphenol oxidase

and tyrosinase. Polyphenol oxidase plays a key physiological role, preventing insects and

microorganisms from attacking or damaging plants, and as part of the wound response of plants

and plant products to insects, microorganisms and bruising. As ripening occurs, fruits and

vegetables are more susceptible to disease and infestation due to the decline in their phenolic

content (Marshall et al. 2000). Phenoloxidase enzymes from fruit and vegetables catalyze the

production of quinines from their phenolic constituents. Later, these quinines go through

polymerization reactions, producing melanins, which exhibit antifungal and antibacterial activity

and help keep fruit and vegetables physiologically healthy (Marshall et al. 2000).

In plants, polyphenol oxidase appears to be localized in the plastids and chloroplasts. It is

also present in the cytoplasm of ripening or senescing plants (Vaughn and Duke, 1984).

Enzymatic browning of fruit and vegetable tissue is catalyzed through the oxidation of

polyphenolic substrates, which are located in the vacuole or cytoplasm, and the enzyme

polyphenol oxidase (PPO), mainly located in the plastids (Barrett et al. 1991) and chloroplasts

(Vamos-Vigyazo, 1981). Compartmentalization prevents enzymatic browning reactions in the

presence of oxygen (Barrett et al. 1991). Mechanical injury or stress (chilling injury, drought,

senescence, or overexposure to O2 or CO2) weakens cell membranes and causes

decompartmentalization, facilitating enzymatic browning.

Some researchers have proposed that changes in cell membrane permeability and release of

either stored substrates from the vacuole or bound enzymes from organelles could be involved in

the enzymatic browning of the tissue. Harel et al. (1964) were first to report an increase in

25

soluble PPO activity during apple ripening. Walker and Wilson (1975); Stelzig et al. (1972);

Vamos-Vigyazo et al. (1985b) noted the existence of solubles and membrane-bound forms of the

enzyme. Barrett et al. (1955) cited by Vamos-Vigyazo et al. (1985) state that there is a higher

content of such solubles as ripening and senescence progress.

Polyphenol oxidase catalyses the initial step in the polymerization of phenolics to produce

quinines, which produce dark colors in the form of melanins (Marshall et al. 2000). These

melanins form barriers and have antimicrobial properties that help prevent the spread of infection

or bruising in plant tissues. Plants that have a high resistance to climatic stress have higher

polyphenol oxidase levels than susceptible varieties. Other enzyme systems in plants, such as

chitinase, peroxidase, lipoxygenase, phenylalanine ammonia lyase, and β-1,3-glucanase show an

increased activity when subjected to mechanical stress. Phenolic compounds are common in

plants and are considered to be secondary metabolites. The polyphenolic composition of fruits

varies depending on the species, cultivar, and maturity level and environmental conditions during

growth and storage. Phenolics contribute to color, bitterness, astringency, and flavor of fruit.

Catechins, cinnamic acid esters, 3-4-dihydroxy phenylalanie (DOPA), and tyrosine are the most

important natural substrates of polyphenol oxidase in fruits and vegetables. Very few of the

phenolic comopounds in fruit and vegetables act as substrates for polyphenol oxidase. In apples,

the common phenolic compounds are chlorogenic acid (flesh), catechin (peel), caffeic acid, 3,4-

dihydroxyphenylalanine (DOPA), 3,a-dihydroxy benzoic acid, p-cresol, 4-methyl catechol,

leucocyanidin, p-coumaric acids and flavonol glycosides. The substrate specificity of polyphenol

oxides varies depending on the enzyme source. Phenolic compounds and polyphenol oxidase are

usually responsible for enzymatic browning reactions in damaged fruits during postharvest

handling and processing. The relationship of the rate of browning to phenolic content and

26

polyphenol oxidase activity has been found in many fruits, like apples (Coseting and Lee, 1987).

In addition to serving as polyphenol oxidase substrates, phenolic compounds serve as inhibitors

of polyphenol oxidases. Cinnamic acids in apples act as substrate analogues and serve as good

inhibitors of polyphenol oxidase (Walker, 1995).

In apple cell vacuoles, phenolic compounds may come into contact with catechol oxidase in

the plastids due to mechanical damage, leading to polymerized quinines and dark colored

compounds (Robitaille and Janick, 1973; Lougheed and Franklin, 1974; Coseting and Lee, 1987;

Klein, 1987; Amiont et al. 1992; Samin and Banks, 1993a; Knee and Miller, 2002). Enzymatic

browning does not occur in intact plant cells because phenolic compounds in cell vacuoles are

separated from the polyphenol oxidase that is present in the cytoplasm. When the tissue is

damaged by slicing, cutting or pulping, the formation of brown pigments occurs, and the

organoleptic and biochemical characteristics of the fruit are altered (Marshall et al. 2000).

Due to the complexity of the substrates and reaction conditions, attempts to relate browning

potential of apples to enzyme activity and phenolic compounds have been inconclusive (Coseting

and Lee, 1987; Klein, 1987). Amiot et al. (1992) analyzed the changes in phenolic compounds

in relation to color changes in bruised tissue of 11 apple cultivars and studied color changes of

damaged apple tissue over time (Samin and Banks,1993b).

27

2.5. Factors Affecting Bruising

Susceptibility to mechanical damage can vary according to various factors (Kays, 1997).

Pre-harvest factors that influence impact sensitivity include hydration level, temperature,

cultivar, species, production practices, turgor, climate, maturity, size, shape, and stress level of

the fruit during growing season (Hyde et al. 1997). In fruit, maturity is also an important factor

because most fruit goes through changes as ripening occurs including flesh softening which

raises the potential for damage due to mechanical injuries. In some cases fruit is harvested before

it is fully ripened to reduce damage due to advanced maturity (Hung, 1993).

As for post-harvest bruising damage, several authors have studied the nature of bruising

and the factors that affect its development. Some of these factors are fruit characteristics,

temperature, packing methods, bin types, transportation, sorting, harvest dates, and storage

conditions (Van Lancker, 1979; Aoyagi and Makino, 1981; Ericsson, 1989; Armstrong et al.

1992). Many studies report various aspects of bruising in apples, e.g. Hyde and Ingle (1968),

Salveit (1984), Thomson et al. (1996), Bollen (2005), Bollen et al. (1999, 2001a, 2001b) and

Toivonen et al. (2007), and many others. However, bruising continues to be a major problem.

Some apple cultivars are more susceptible than others (Hyde and Ingle, 1968; Brusewitz and

Bartsch, 1989; Hyde and Zhang, 1992; Ericsoon and Tahir, 1996; Hyde et al. 1997; Kupferman,

2006).

Environmental factors can also influence susceptibility to damage. Temperature, oxygen

concentration, and relative humidity can affect fruit when a mechanical stress is applied. This

may be due to a direct effect on the mechanical properties of the fruit, or to the development of

the injury after the damage has occurred.

28

2.5.1. Turgor Pressure

Research regarding the influence of apple turgidity on bruising indicates fruit suffers less

bruising on impact if the turgor is reduced by weight loss. This is due to the relation of turgor

pressure to brittleness of the tissue. Highly turgid fruit is more susceptible to impact damage than

fruit at lower turgor (Johnson and Dover 1990; Banks and Joseph 1991). This was also reported

for peaches (Horsfield et al., 1972; Brusewitz et al., 1991), apples (Garcia and Barreiro, 1995),

and bananas (Banks and Joseph, 1991).

Water content is related to the turgor pressure (Zhang, et al.,1992). It is also an easier

factor to measure than turgor pressure. Zhang, et al. (1992) analyzed the effects of humidity and

temperature on bruise susceptibility. At 50% relative humidity, stored apples exhibited lower

bruise susceptibility than at 75% and 100% relative humidity. Warming the apples to 60°F

before handling also reduced bruise susceptibility. ‘Red Delicious’ apples were more bruise

susceptible than ‘Golden Delicious’ apples under the same conditions. Such results show that

temperature and storage humidity just before handling are important in reducing the bruise

susceptibility of ‘Red Delicious’apples and ‘Golden Delicious’ apples. New Zealand growers

observed that apples were more susceptible to bruising when picked early in the morning, at

lower temperatures, or after a long period of rain since apples would have a higher turgor due to

water intake (Samin and Banks, 1993). Viljoen et al. (1996) recommended handling fruit

carefully early in the morning and picking more slowly until humidity dropped and temperatures

rose.

In peaches, the turgor effect is not large enough to offset the increase in bruise

susceptibility. Peaches soften during storage and ripening while apples soften less, but a small

increase in bruise susceptibility could be offset by the effect of changes in turgor. Knee and

29

Miller (2002) mentions that this could explain the inconsistencies in the literature with respect to

changes in bruise susceptibility during storage of apples. Inducing weight loss before handling

has been suggested to reduce bruise damage in ‘Bramley’s seedling’ apples (Johnson and Dover,

1990).

Previous research has dealt with a small amount of stress and strain to reduce failure of

fresh apple tissue with higher turgor (Konstankiewicz and Zdunek, 2001; Scanlon et al. 1996).

This is due to an increase in the initial tension of the cell walls by increase of turgor. When tissue

is compressed, the intercellular pressure increases with the tension in the cell walls, which causes

cracking at smaller stresses (Konstankiewicz and Zdunek, 2001). This behavior was related to

cell rupture primarily due to failure mode (Lin and Pitt, 1986).

2.5.2. Firmness

Fruit firmness has been related to bruise susceptibility (Brett and Waldron, 1990).

Softening (loss of firmness) is one of the most significant changes associated with ripening of

fleshy fruits (Kays, 1997). Alterations in texture affect edibility of the fruit and the length of time

the fruit may be stored. In some fruit, softening is important in developing optimum quality.

Maximizing the control over textural changes, preventing, synchronizing, or accelerating them,

is of great concern to the fresh food industry. Acceptable flesh texture has a narrow range that

can be easily exceeded, reducing the quality of the product. With the exception of some turgor-

mediated textural alterations, softening in most fruits represents an irreversible process.

30

Softening is part of fruit ripening and is due to changes in the cell walls and wall

degradation. The enzyme in charge of this process is polygalacturonase. Enzyme activity

increases during fruit softening in peaches, pears, avocados, dates and tomatoes. During ripening

there is a rise in ethylene production in the fruit which triggers polygacturonase production. The

enzyme degrades the middle lamella of parenchyma cells which weakens cell-cell adhesion

(Brett and Waldron, 1990). The degree of firmness has been related to bruise susceptibility.

Ragni and Berardinelli (2001) mentioned that fruit with a high firmness (measured with Magnes-

Taylor) resulted in high bruise susceptibility, this could be due to the positive correlation

between Magness-Taylor firmness and apple stiffness, and the positive correlation between apple

stiffness and bruise susceptibility (Van Zeebroeck, 2005 cited by Van Zeebroeck et al. 2007; Van

Zeebroeck et al. 2007). Contrary to this, Hyde an Ingle (1968) observed that in some cultivars

bruise susceptibility is not related to pulp firmness. This contradicts what other authors mention

about delayed harvest, as maturity increases bruise susceptibility increases (Brusewitz et al.

1991; Diener et al. 1979; Klein 1987; Kvaale et al. 1968; Salveit 1984).

Irrigation schedules in the last weeks before harvest have been shown to influence fruit

firmness. Watered trees produced firmer fruit than non-irrigated trees. The rate of fruit ripening

was shown to be unaffected. No changes were detected in skin physical properties or bruise

susceptibility in relation to irrigation schedules. The values of deformation at skin puncture were

in every case lower than 0.7mm at harvest, and higher than this value after storage in ‘Golden

Supreme’ and ‘‘Golden Delicious’’ apples. Analysis indicated a relationship between

deformation at skin puncture (DSP) and bruise susceptibility. Fruits with low values of

deformation skin puncture (DSP) in turgid fruits showed high values of bruise volume,

particularly in ‘Golden Delicious’ apples. Turgid fruits show different impact response compared

31

to less turgid fruits. The difference in turgidity can also explain why fruit at harvest were more

susceptible to bruising than fruit after a period of storage as tested by other researchers.

Stored apples do not react the same as fresh picked apples due to the time and

temperature at which apples are stored. Bruise volume increases at high bruising and storage

temperatures (Salveit, 1984). This due to the possible increase in enzyme activity responsible of

browning in injured tissue. Therefore, bruising can be cultivar related as well as the freshness of

the fruit (just harvested or previously stored). If temperature has no relation with bruising

volume, it does have a great influence on fruit development. Warm weather during the first six

weeks of fruit development makes softer fruit at harvest, and firmness also decreases more

rapidly before harvest (Trompt, 1995 cited by Viljoen et al. 1996).

2.5.3. Maturity

Fruit is considered mature when it meets its requirements for harvest (harvestable

maturity). Harvesting at an optimum stage of maturity is important to obtain good quality and to

maintain this quality after harvest (storage). Harvest based on calendar dates is the easiest way

to schedule harvest when the growing conditions are consistent with previous years. However,

blooming dates vary with pre-bloom temperatures year to year (See Table 3). The rate of fruit

maturation also depends on the temperature during the growing season (Eggert, 1960;

Warrington et al. 1999). Studies show that optimum harvest dates can be predicted based on the

date of 50% full bloom and summer temperatures (Luton and Hamer, 1983). The duration of

32

harvest depends on cultivar and environmental conditions that have an effect on the time and

length of the harvest maturity.

For growers harvesting the fruit at the right stage of maturity is a function of the

product’s properties, but mainly of the market demands (Kays, 1997) (Figure 4). Fruit harvesting

dates depend mainly on the market demands more than the optimum maturity of the fruit.

Fruit maturity and harvest dates have an effect on bruise damage. Saltveit (1984) and

Klein (1987) also noticed that delayed harvest enhanced fruit sensitivity and this effect was also

cultivar-dependent. Klein (1987) found that susceptibility damage increased by late harvest. He

also found that bruising increased by delaying harvest; an increase in cell sugar content as

maturity progressed caused the cell turgidity to increase. Hyde and Ingle (1968) observed that

fruit from the second and third harvest dates presented on average larger bruises than fruits from

the first harvest (5 day intervals). Contrary to the above, Studman et al. (1997) and Pang et al.

(1992) observed that the greener side of the apple was more susceptible to bruising compared to

the red side of the apple (more mature side). Brusewitz et al. (1991) separated harvested peaches

into three ripeness categories and found an increase in bruise volume with ripeness. In apples

Diener et al. (1979) reported a 30% decrease in bruise volume for ‘Golden Delicious’ apples

within a harvest period of 27 days. Klein (1987) observed a 10% increase in bruise volume in

apples harvested over a period of 30 days.

After tissue is damaged, its visual properties may change due to the tissue maturity. It is

important to note that discoloration of a bruise tends to disappear over time due to re-absorption

of the cell sap in a water soaked tissue (Radossevich et al. 1968), but the development of this

discoloration is variable (Radossevich et al. 1968; Kvaale et al. 1968). This variability is

correlated to the physiological stage of the fruit. Visual darkening of the tissue that resulted from

33

water soaked tissue was temporary (areas adjacent to the damage area), but darkening due to

browning was permanent in the damaged area. If the same pressure is applied in a bruised area,

the size of the bruised area will increase depending on the ripeness of the fruit (or harvest time)

(Kvaale et al. 1968). Kvaale et al. (1968) showed that the discoloration of bruises made after

storage did not disappear with time and there was visible flesh browning, but mature green fruit

did not produce the intense tissue browning as ripe yellow fruit. The time of harvest can affect

the way tissue reacts to bruising. Maturity index is correlated to bruise depth and pulp firmness

(Dedolph and Austin, 1962; Mohsenin et al. 1962). The difference in bruise susceptibility is

related to physiological and biochemical properties of the tissue that can be affected by cultivar,

maturity, and storage time of apples.

Ethylene is a natural product of ripening fruits (Gane, 1934). Ethylene acts in apples as a

natural hormone triggering mechanism for the induction of respiration of climacteric fruit.

Ethylene may trigger a ripening response inducing earlier ripening in fruit. It has been thought

that bruising can increase ethylene production (Robitaille and Janick, 1973). As fruit approaches

its physiological maturity, there is an increase in ethylene production. Previous research shows

that different types of wounds such as slicing vs. maceration have different physiological

responses. Slicing increased respiration rate (Burg and Thimann, 1960; Dedolph and Austin,

1962), but the response of apple tissue is variable and depends on the cultivar, morphology, size,

and maturity of the fruit, and permeability of the peel to gases (Burg, 1962; Burg and Thimann,

1959; Burg and Thimann, 1960). In pear (Dedolph and Austin, 1962) and tomato (Eaks, 1961)

slicing caused an increased production of ethylene and other volatiles from fruit tissue, but

ethylene production in apple slices or plugs was reported to remain unchanged (Eaks, 1961), or

to decrease in proportion to the size of the cut surface (Gaston and Levin, 1951). Eaks (1961)

34

found that maceration of apple tissue reduced ethylene production. The crushing of the cells

could increase the activity of the oxidative enzymes and lower the O2 concentration under the

damaged skin, depressing ethylene synthesis. Robitaille and Janick (1973) had surprisingly

different results than expected: neither wounding nor bruising increased ethylene production.

They observed that bruising decreased ethylene production in apples due to the damage done to

the cells which were responsible for ethylene production.

2.5.4. Harvest and Post-harvest factors

Bruises caused by the picker’s thumb pressure have not been widely studied. The picker

is the main cause of bruise damage during hand harvesting operations (picking, bin filling, bin

hauling) (Brown et al. 1993). The pickers influence bruising until the apple is in the bin. Shulte

et al. (1991) did some tests on the damage caused by the pickers and concluded that the main

factor affecting bruising in apples was the way pickers harvested the fruit.

There are several techniques to reduce picking bruises. The use of gloves and short

fingernails helps reduce punctures (Shulte et al. 1991). Contrary to expectation, the use of gloves

(soft/silky white cloth) to pick apples resulted in a significant 5.7% increase in bruising compare

picking with no gloves (Brown et al. 1993). According to Brown et al. what reduced bruising

were a cushioned bucket for picking, supervision, daily quality inspections of the harvested fruit,

and adequate instruction on how to correctly pick and handle apples. These were the most

important factors for minimizing fruit damage while picking. A well-trained and supervised

picker may cause an average of 1.0 bruise/apple, but an untrained and unsupervised picker can

35

triple the amount of bruises. The use of picking bags with rigid sides can prevent bruising when

leaning against the container. Care can be taken when placing the apples in the container, instead

of violently dropping them. Lastly, once the picking bag has been filled, the picker should slowly

lower the bag into the bin and proceed to release the apples from the bag in a flowing motion so

the apples will flow out of the bag instead of hitting each other. This will also reduce the vertical

drop of the apples into the bin (Fisher 1942; Gaston and Levin, 1951). Schomer (1957) also

found that most harvest damage is due to dropping rather than placing fruit into picking

containers, and the careless filling of bins. Shulte et al. (1991) confirmed that hand harvesting of

apples into picking bags and its placement into the bin is an important source of apple damage.

The pickers’ ability to pick and handle fruit carefully, without any instruction, was the

most significant factor in apple damage during hand harvest and placement into bins. In Shulte

et al. (1991) states the picker was the most important factor in determining the amount of

bruising accumulated during hand harvest and placement of ‘Golden Delicious’ apples into bulk

storage bins. Despite the different techniques used, growers are doing their best to reduce apple

bruising in the field, but bruising still affects 20% of harvested apples (Sargent et al. 1987).

2.5.4.1. Evaluating Pickers’ Bruises Based on Bruise Color

When bruising a fruit, its evaluation is important to understand the damage made.

Grading impacts the amount of money the growers get paid for their fruit. Bruise size is

important in a damaged area, but bruise evaluation is also influenced by the intensity of the

discoloration due to its visibility. According to Samin and Banks (1993) discoloration of apple’s

36

bruises develops with time, but fades as water and dry matter are lost from the bruised area. The

changes in discoloration might be affected by the tissue’s water status; which might also be

influenced by the surrounding tissue (healthy tissue) that could absorb material from the

damaged area. The bruise could reduce its turgor by decreasing local water potential. The water

potential is related to cell sap released from the damaged area. Increases in hue angle with time

indicated a shift from brown towards yellow. Samin and Banks (1993) observed changes

associated with a visible lightening and drying of the bruises from the second day after bruise

application as surrounding healthy tissue absorbed the sap. The reduction of dry matter loss

compared to the fresh weight could mean that the mobility of the dry matter decreased with the

low amount of water content of the bruised tissue.

Most of the discoloration in bruised tissue occurs within a few hours of the impact

(Samin and Banks, 1993). The bruised tissue rapidly turns darker (decreased L value), becomes

browner (decreased H), and increases in color intensity (increased C). This could be due to the

oxidation of phenolics and soaking of the tissue. An increased discoloration intensity was

observed between 10 and 24 hours; after that, the changes were gradual and if longer, the results

would be less obvious to detect the damage due to the fading of the bruised area. Contrary,

Varith et al. (2003) found that 48 hours was a good parameter to determine bruise damage in

apples.

Bruise visibility varies among cultivars depending on the fruit’s skin color (Toivonen et al.

2007; Zhang et al. 1992). Apple cultivars with a dark skin like ‘Red delicious’ cover up the

bruise more than lighter cultivars like ‘Golden delicious’ apples.

37

2.5.5. Storage and Temperature

Storage conditions may prolong the postharvest life of fruits and vegetables, but cannot

stop the senescence (Kvaale et al. 1968). Apple behavior is not the same when harvested fresh

compared to fruit held for months in cold storage. Fruits and vegetables get physiologically older

in storage. The main objective of storage is to slow down the ripening /aging process. Kvaale et

al. (1968) reported that ‘Golden Delicious’ apples bruised differently at harvest compared to fruit

stored for a period of time. The bruise sensitivity and development was related to ripening and

senescing of the harvested or stored apple. Kvaale et al. (1968) concluded that ‘Golden

Delicious’ apples can be handled at harvest and just after harvest without significant permanent

discoloration from small bruises. After harvest and storage, there is a change that results in

permanent discoloration in the damaged tissue. After storage, there is no discoloration recovery,

which means that the bruise can still be visually detected. It intensifies and a larger indentation

results from a small pressure. Even though the clearing of a dark discoloration is noticeable,

permanent damage has occurred and tissue in the bruised area has been killed, resulting in a soft

spot or skin depression. The clearing of the discoloration only occurs in fresh fruit after harvest

when the fruit is physiologically young. Clearing does not occur in physiologically older fruit,

stored fruit.

Contrary to prior findings, Menesatti et al. (2003) reported very high bruising

susceptibility of ‘Golden Delicious’ apples at harvest time. After five months of controlled

atmosphere (CA) storage, bruising resistance increased 4-6 times, but after 5 to 8 months,

resistance decreased (approximately 18%). The storage, and to a lesser degree the conditioning

following CA storage, determined an important modification of the fruit tissue, which became

38

softer, but more elastic. Weight loss causes a significant reduction of the cell turgor. This elastic

behavior allowed the fruit to be more resistant to impact bruising. Storage treatment following

the applied load causing a bruise reduced bruising percentage by 95% at harvest, 37% after 5

months of storage, and 21% after 8 months of storage. [Menesatti et al. (2003) also emphasize

that the storage time, and the conditioning after CA, are important in modifying the fruit tissue

behavior. The rheological behavior of fruit is to become softer and more elastic. This may be the

result of weight loss that reduces cell turgor, allowing the fruit to be more elastic in response to

the impact damage which would cause a bruise.

Fruit tissue strength can be influenced by temperature and hydration (turgor). Colder and

more turgid fruit and vegetable tissues are more brittle and sensitive to impacts, and are easily

bruised. Hydration conditioning in apples can reduce bruising problems during handling (Hyde,

1997). Fruit temperature when bruising is also important (Saltveit, 1984; Thomson et al. 1996;

Baritelle and Hyde, 2001). Saltveit (1984) and Thomson et al. (1996) demonstrated that storage

temperature after bruising affects bruise development.

Saltveit (1984) pointed out that in apples, higher fruit temperature at impact increased

bruise volume. He also observed more bruise volume by increasing storage temperature and

suggested it could be related to increased enzyme activities. He found a reduction in bruise

volume in apples injured at lower temperatures regardless of the holding temperature. Bruising

was also reduced in apples held at low temperatures, regardless of the temperature during

bruising. Contrary to Saltveit’s idea, other researchers reported that higher temperatures during

impact reduced bruise susceptibility (Schoorl and Holt, 1977; Van Lacker, 1979;

Tsukamoto,1981) whereas Klein (1987) did not find any relationship between temperatures at

impact and bruise susceptibility.

39

Resistance of fruit to bruising is reduced with delayed harvest has been observed by other

reaserchers (Aoyagi and Makino, 1981; Lidster and Tung, 1980; Klein, 1987). Lidster and Tung

(1980) found no correlation of resistance to bruising due to fruit density, firmness, size or

polyphenol content. Ericsson and Tahir (1996a) supported Saltveit’s (1984) findings with respect

to pre-cooling and bruising, depending on the cultivar. The decrease of bruise weight percentage

(BWP) in pre-cooled sensitive cultivars may be caused by the inhibition of the activity of

enzymes involved in browning of damaged tissues (Maness et al. 1992). Many authors report

that impact at high temperature promoted fruit resistivity due to a decrease in moisture (Schoorl

and Holt, 1977). Klein (1987) explained that the vapor pressure gradient between the atmosphere

of the cold room and the air spaces in the fruit cortex resulted in moisture loss from the fruit and

a decrease in cell turgor. As turgidity decreases, the susceptibility to bruising decreases. Ericsson

and Tahir (1996b) found that precooling by forced air decreased bruising due to moisture loss.

Temperature has a small influence on bruise susceptibility, but dehydration (by losing 1-

2% of weight) can reduce bruise damage up to 50% (Hyde et al.,1997). More turgid fruit will

have poorer resistance and larger bruises than those with less turgidity after dehydration. Some

factors that influence impact sensitivity are: hydration level, temperature, cultivar, production

practices, climate, maturity, size and shape. Each factor will vary depending on the variety. The

influence of production practices includes the amount of water and nutrient stress, and the stress

during the growing season.

Reducing hydration levels dramatically reduces bruising for both red and ‘Golden

Delicious’ apples (Zhang, 1994). Bruising is reduced by 50% by reducing hydration level

increasing weight loss only 2%. Red delicious apples has a lower bruise threshold than ‘Golden

Delicious’ apples. This is consistent with Schulte et al. (1992). According to Zhang (1994)

40

temperature has no significant effect on bruise resistance for red delicious or ‘Golden Delicious’

apples. These results indicate that in order to reduce bruising of ‘Golden Delicious’ apples

coming out of CA storage in February, warming is not as important as reducing hydration

(turgidity). Slight dehydration of the fruit will reduce bruising by improving bruise threshold.

Adjusting fruit turgidity (hydration) and temperature can improve bruise threshold (Hyde

et al., 2001). For apples, turgor has more effect than temperature on bruising, as for Barlett pears

turgor and temperature are important. In apples, a slight reduction in hydration of 2 to 3% mass

loss doubled the bruise threshold. Hyde et al. (2001) concluded that bruise threshold improved

with moisture loss, but temperature had only a small effect at lower turgor level. They concluded

that bruise threshold and bruising could be controlled by managing the fruit’s hydration level,

which influences turgor pressure.

2.6. Introduction to Microscopy

Research about bruising has been difficult to apply commercially in the apple industry

(Saltveit, 1984; Brusewitz and Bartsch, 1989; Thomson et al. 1996; Baritelle and Hyde, 2001).

Generating data useful for the management of bruising involves recreating real injuries that

apples suffer during harvest and handling. It is important to be able to distinguish among types of

damage. Compression damage would be by simulating harvest bruises or impact damage

simulating packing line bruises. Compressive and impact injuries have different effects on the

fruit tissue (Banks et al. 1991) should be studied more to reduce bruising in the industry

41

(Toivonen et al., 2007) and, even though bruising is of great economic significance, there have

been few physiological studies on this subject.

According to Pitt (1982), Pitt and Chen (1983) Holt and Schoorl (1982, 1983b, 1984b), Van

Woensel and De Baerdemaeker (1983), Lin and Pitt (1986), Vincent (1990), Gao and Pitt (1991)

Roudot et al. (1991), Khan and Vincent (1990, 1993ab) Abbott and Lu (1996) Loodts et al.

(2006) bruising is a consequence of breakage of intercellular bonds, propagation of cell wall

ruptures, or cell deflation as a result of loss and diffusion of cell fluid. These conclusions resulted

from analyses at a cellular level, modeling cell mechanical response to external loads. Most

research related to failure mechanisms has been done with quasi-static compression

(thermodynamic reversible process under equilibrium). Many models have attempted to explain

the response of fruit tissue under the application of external loads and strains (Chen et al. 1986;

Dal Fabro et al. 1980; Holt and Schoorl, 1977b; Nilsson et al. 1958; Pitt, 1982; Segerlind and

Dal Fabro, 1978), but none of these investigations have been based on the microscopic anatomy

of cells.

The use of different microscope techniques has help understand tissue organization and

cells and in this study for bruised tissue. The use of different techniques for tissue preparation

and analysis is part of the microscopy techniques. The microscope in general has been a useful

tool to solve and understand many biological problems. Every technique provides a piece of

information that is useful for research. The use of different microscope techniques can help

understand what occurs in bruised tissue. Experimental methodologies for studying in situ

deformation are limited. Therefore, using all the possible techniques might give a better

prospective of what is happening in the damaged tissue.

42

Optical microscopy does not have enough resolution or depth of focus to study cell

separation and breakage (Donald et al. 2003). Using scanning electron microscopy (SEM) solves

these problems, but the tissue is not studied in its natural state due to the procedure used to

preserve the tissue, such as high vacuum for dehydration and coating with a conductive layer of

gold. Images obtained with SEM might not be representative of the natural structure of the

hydrated ‘live’ specimen. Pierzynowska-Korniak et al. (2002) used the scanning microscope to

determine difference in cell structure in different apples cultivars. With this they could determine

that different cultivars have unique cellular structures

Confocal scanning laser microscopy (CSLM) has been used to analyze fracture properties

of stored and fresh ‘Granny smith’ apples (Alvarez et al. 2000). The advantages of CSLM over

conventional microscopic techniques are that the material is viewed fresh and the structure may

be viewed at different depths (see Figure 5c). Using CSLM images, they confirmed that storage

resulted in a reduction of cell to cell adhesion; internal air space increased during storage of

‘Granny smith’, and apples tasted mealy when the intercellular air spaces were approximately

20% (Tu et al. 1996). Large intercellular spaces in apples are due to degradation of the middle

lamella and a reduction in cell adhesion. CSLM is a good measure of deterioration of apples

during storage. Funebo et al. (2000) also used CSLM to observe apple cells after dehydration and

rehydration processes and observed cell separation and disruption of cell walls (see Fig. 6).

Veraverbeke et al. (2001) used various techniques such as CLSM, ESEM and SEM to analyze

wax layers on apples. They observed that images under SEM showed more damaged surface due

to the destructive character of the technique. The images with ESEM and CLSM do not show

flaked and shallow micro-cracks forming an interconnected network on the surface because of

the use of fresh material, not processed. Using the environmental scanning electron microscope

43

(ESEM) has overcome these problems. ESEM requires only minimal sample preparation, just by

cutting samples the right size to fit in the stubs (holders).

The advantage of some microscopy techniques is that deformation of cells can be

observed while applying a force as Oey et al. (2007) did with apple cells. Glenn and Poovaiah

(1987) used light microscopy, scanning electron microscopy, and transmission electron

microscopy to show that ‘Golden Delicious’ apples had high textural quality at harvest time due

to cell-to-cell contact, as compared to apples stored for 7 months (see Figure 7). The use of light

microscopy helped Ormerod et al. (2004) observe cell rupture and separation in carrot tissue and

chinese water chestnuts. With these results they could understand the mechanism; structure and

failure properties of the tissue (see Figure 8). Hallett et al. (2005) used light microscopy to

examine changes in cell wall due to ripening in kiwi fruit.

The use of different microscopy techniques provide a unique array of information that

other techniques cannot or might lack. It is like a jigsaw puzzle, putting the pieces together in

order to build a larger image of what is going on in the tissue or cells, in order to understand it.

44

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Tables and Figures

Table 1. Frequency of disorders and diseases reported in USDA inspections of apple

shipments

(Crisosto et al. 1993)

Apple bruising incidence during harvest and postharvest handling

Bruises/fruit % bruised fruit

Picking/dumping 2.6 18.8

Hauling 2.2 15.9

Packing line 5.5 61.6

Transportation 0.5 3.78

Total 13.8 100

Table 2: Other definitions of a bruise:

Mohsenin et al. (1962) defined a bruise as cell injury resulting from the application of force (or pressure) and causing flesh browning. The inclusion of flesh browning in the definition is more for easily locating the bruised area than typifying either the cause or response. a) Application of very slight pressure results in elastic deformation and apparently no injury. b) Application of greater pressure results in plastic deformation of the tissue and damaged or burst cells. c) Application of further pressure results in the additional rupture of the epidermis. Chen et al. (1986) direct visual presence of discoloration or fractures. Holt and Schoorl (1977) bruising is caused by cell bursting when the flesh is compressed. Diehl et al. (1979) and Pitt (1982) cell rupture occurs due to mechanical action. Ericsson and Tahir (1996) injured area is flattened, soft, brown and has a high respiration rate. Blahovec (1999) dark spot. Robitaille and Janick (1973) a bruise can be considered to be cell rupture and flesh browning from force application. Crisosto et al. (1993) a bruise usually appears as an indentation on the surface and brown discoloration of the flesh underneath.

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Table 3. Average maturity index for some apples and pear cultivar at optimum maturity stage (Jackson, 2003).

Cultivar DFFB

(days)

Firmness

(kg)

Skin

color

TSS

(%)

Titratable

acid

(g/100g)

Starch % Seed

color

‘Golden

Delicious’

131 7.63 2.3 12.9 0.54 18 5

Figure 1. Tissue elements of Malus (apple) fruit (Esau, 1965).

Tissue elements of Malus (apple) fruit. A,B, epidermis and subjacent collenchymatic tissue from young (a) and mature (b) fruits. C,D, parenchyma of the floral-tube part of the flesh. C was taken closer to the surface, D farther away. E, parenchyma from exocarp. F,G, endocarp from young (f) and mature (G) fruits. A,C-E, from transections of a fruit 1 cm in diameter; F, from radial

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Radial long sections from the equator of the fruit cheek showing the skin structure at different days from blossoming of (a) a russet (‘Brownless Russset’) and (b) a normal (‘Cox’s Orange Pippin’) cultivar. H, hair base; Cu, cuticle; E, epidermis; Co, collenchymas or hypodermis; A, airspace; Pm, phellem or cork; Pg, phellogen; Pd, phelloderm.

Figure 3. Radial long sections from equator of the fruit cheek showing the skin structure at different days from blossoming (Skene, 1962).

Structure of a mature apple fruit. (a) Vertical section; (b)

Figure 2. Structure of a mature apple fruit (Robbins, 1933).

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Figure 4. The timing of harvestable maturity within the normal developmental cycle of a plant varies widely between individual products (Watada et al. 1984).

The timing of harvestable maturity within the normal developmental cycle of a plant varies widely between individual products. Some (e.g., bean sprouts) have just begun development when they reach a harvestable maturity whereas others (e.g., grains, seeds) complete the cycle from seed to seed.

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Figure 5. Confocal scanning laser images of fresh and osmotically manipulated Granny Smith (Alvarez et al. 2000).

Confocal scanning laser images of fresh and osmotically manipulated Granny smith tissue with storage time a,b, at day 0 and c, d after 28 days in shelf-life storage.

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Figure 7. Light micrographs of flesh from untreated and calcium treated ‘Golden Delicious’ apples (Glenn and Poovaiah, 1987).

Light micrographs of flesh from untreated (A) and calcium-treated (B) ‘Golden Delicious’ apples after 7 months of storage. The untreated fruits were mealy and had spherical cells with very little cell to cell contact (A). The calcium-treated fruits were firm and had polygonal shaped cells due to a high amount of cell to cell contact.

Figure 6. CSLM images of apple cells dehydrated at 80°C at higher magnification (Funebo et al. 2000).

CSLM images of apple cells dehydrated at 80°C at higher magnification than in Fig. 7, without (a) and with (b), microwave heating initially. Apple cells ``blanched'' with microwave energy separate from each other and cell walls are more disrupted, which is shown in (b), compared to apple cells from air dehydration in (a). The white arrows in (b) indicate some of the ruptures in the cell walls.

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Figure 8. A schematic generic model linking the mechanical, structural and failure properties of plant tissues (Janes et al. 2005).

61

CHAPTER TWO

BRUISING PROFILE OF FRESH APPLES ASSOCIATED WITH TISSUE TYPE AND STRUCTURE

K. Mitsuhashi, M. J. Pitts, J. K. Fellman, E. A. Curry and C. D. Clary

The authors are Kay Mitsuhashi, Graduate Student, Carter D. Clary, Ph.D., ASABE Member,

Assistant Professor, John K. Fellman, Professor, Department of Horticulture and Landscape

Architecture, Washington State University, Pullman, Washington, Marvin J. Pitts, Ph.D.,

ASABE Member, Associate Professor, Biological Systems Engineering, Washington State

University, Pullman, Washington and Eric A. Curry, Ph.D., Research Plant Physiologist, U. S.

Department of Agriculture, Agriculture Research Service, Tree Fruit Research Laboratory,

Wenatchee, Washington. Corresponding author: Carter D. Clary, Department of Horticulture

and Landscape Architecture, P.O. Box 646120, Washington State University, Pullman, WA,

99164-6414, phone: 509-335-6647; fax: 509-335-8690; email: cclary@wsu.edu.

ABSTRACT

It is important to understand how apples bruise in order to prevent or reduce bruising. Tissue

from ‘Golden Delicious’ apples was analyzed to determine the bruising mechanism at different

maturity levels. Bruising was induced by an artificial finger attached to an Instron machine

applying an external load to fresh picked ‘Golden Delicious’ apples. To understand the bruising

mechanism involved, we used fluorescence microscopy with Calcofluor fluorescent dye to

identify cell walls and CDFA to identify cell membranes in the bruised and discolored tissue.

Together with SEM, different breakage mechanisms were observed in the bruised area. We

observed that 48 hours following damage, the bruised tissue was comprised of dead and live

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cells, burst, crushed and intact cells. The more intercellular space there was in the tissue, the

more tissue damage occurred. Airspaces were the weakest points in the tissue structure, and

damage initiated in those points. As apples matured, there was an increase in damaged cells

surrounding larger intercellular spaces.

Keywords: apples, bruising, harvesting date

INTRODUCTION

Understanding the mechanisms of bruising at a cellular level in apples is important in

order to help explain visual damage seen by consumers. If the mechanisms are well-known and

understood, damage to apples during harvest could be reduced, benefiting this multi-billion

dollar global industry.

Apple epidermis is comprised of epidermal tissue (the outermost layer of cells), followed

by subjacent collenchymatic tissue known as the hypodermis. Beneath this are the parenchyma

cells (Esau, 1965). The hypodermis may contain up to six layers of collenchyma cells, which

contribute to the strength of the epidermis. The strength of the top cell layers transmits external

force to the underlying parenchyma cells (Knee and Miller, 2002).

Each of the cell type characteristics are key to developing an understanding of bruising in

apples. Collenchyma cells are smaller, thicker-walled, more elastic cells that resist an applied

force better than do parenchyma cells, and are commonly interpreted to be structurally

specialized as supporting tissue (Esau, 1965). Collenchyma cells have specialized, irregularly

thickened, pectin-rich, primary cell walls that function in support of growing parts (Taiz and

Zeiger, 2002). The Greek word colla, glue, refers to the thick, glistening wall characteristic of

collenchyma cells. Collenchyma cells are well-packed with smaller intercellular spaces,

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compared to parenchyma cells which are larger and comparatively thin-walled and are

interspersed with large intercellular spaces. Thick walls and close packing make collenchyma

tissue strong and resistant to compression. Collenchyma tissue combines considerable tensile

strength with flexibility and plasticity.

Parenchyma cells are not as flexible as collenchyma cells. Parenchyma, derived from the

Greek para, beside, and en-chein, to pour, is a combination that depicts a semi liquid substance

“poured beside” other tissues which are formed earlier and are more solid. Parenchyma tissue is

metabolically active tissue, comprised of thin-walled cells, with air-filled spaces at the cell

corners (Taiz and Zeiger, 2002). These intercellular airspaces may be relatively large, which

reduces the amount of cell-to-cell contact (Hulbary, 1944, in Esau), thereby weakening the tissue

(Alvarez et al 2000), resulting in areas vulnerable to damaged by external force.

Holt and Schoorl (1977) proposed a failure mechanism for apples bruising at the cellular

level. Their model shows burst cells in the affected area and distorted cells under the burst cells,

followed by a layer of unaffected cells some distance away from the compressed surface. They

assume all apple cells are parenchyma cells. Holt and Schoorl (1982) mention that bruising is a

mode of failure associated with damaged tissue due to cell bursting. When cells burst, they retain

no mechanical strength. Diehl et al. (1979) observed that cells change shape when an external

load is applied, and the cell walls stretch because cellular content is relatively incompressible.

Cell breakage occurs when the cell wall fractures. Bruising can also be considered a distortion or

shear phenomenon. According to Holt and Schoorl (1982), bruising is a shear phenomenon, like

slip but unlike cracking. Peleg et al. (1976) showed that slip failure occurred in unripe mango

and papaya, while bruising occurred in ripened fruit.

Alvarez et al. (2000b) emphasize the basic principle of fracture mechanics in all solids is

inhomogeneities. Irregularities in tissue structure essentially weaken the solid and make it more

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vulnerable to damage. The strength of a material is therefore directly related to the magnitude

and distribution of these irregularities. Fractures begin at the site of such irregularities and grow

and traverse the solid, creating a new fracture surface. Alvarez et al. (2000) tested various

tissues like carrots, celery, cucumber and apples under the same external load. Their results

showed that tissues with small cellular structures sustain the least damage. Carrots had the

toughest tissue due to their small parenchyma cells and lack of intercellular spaces. Cells are in

intimate contact with neighboring cells, and the degree of adhesion is higher, resulting in fewer

flaws or inhomogeneities. Such flaws can act as stress concentrates and promote premature

failure. In cucumber and apples, and less in celery, the cellular structure contains more

intercellular spaces and the cells are not in intimate contact. There are many large sites where

the shapes of the cells prevent adjacent cell walls from touching. Later in fruit development,

depolymerisation of the pectic polysaccharides occurs through ripening, further reducing cell

adhesion.

Apples are anisotropic, not homogeneous in all directions, with a radially oriented

network of airspaces (Khan and Vincent, 1990). This network of airspaces is known to be the

weakest area in apple fruit tissue (Vincent et al. 1991). According to Harker et al. (1997), the

actual mechanism of breakage is characterized by three modes of failure: cell fracture, rupture

and cell-to-cell debonding. Each cell exhibits one of these failure modes. Fracturing is

characterized by cells cleaving across the equator; rupture by cell bursting and collapse; and cell-

to-cell debonding by the separation of cells with no damage or only minor deflation or distortion,

while other cells remain intact.

Harker et al. (2006) observed that firmer fruit showed fractured and ruptured cells, while

softer fruit showed cellular debonding when the tissue was externally damaged. In stored fruit,

the failure pattern depended on the firmness of the fruit. Fruit that softens during storage shows

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both a fracture surface and intact cells, due to cell-to-cell debonding. Firm stored apples had the

same characteristic as firm apples after one day of storage.

A final contributor to bruising in apples is oxidative browning, a result of the breakdown

of membranes in cells of plant tissue (Toivonen and Stan, 2004). When physical stress or

deteriorative process occurs, such as wound response or senescence, is initiated and

compartmentalization of cells begins to fail (Marangoni et al. 1996). The result is the mixing of

polyphenol substrates like catechin or polyphenols with polyphenol oxidase and/or phenol

peroxidase (Degl’Innocenti et al. 2005). Toivonen and Brummell (2008) point out that

membrane stability is a major factor controlling the rate of browning. Ascorbate in the tissues

can work to inhibit browning because it is a universal antioxidant (Noctor and Foyer, 1998) and

can quench lipid alkoxyl and peroxyl radicals involved in membrane deterioration (Espin et al.

2000b).

In riper fruit more bruise damage has been observed. Brusewitz et al. (1991) found an

increase in bruise volume with ripeness. Kvaale et al. (1968) separated ‘Golden Delicious’

apples into two main groups based on skin color (green vs. yellow) which represented different

stages of maturity of apples for the fresh market. Saltveit (1984) and Klein (1987) also noticed

that delayed harvest enhanced fruit sensitivity to bruising. Based on previous research there is a

difference in bruise sensitivity, but understanding what caused those differences is one of the

objectives of this work. Previous works have focused and measured the damaged due to an

external load at different maturity stages, but have not been explained or proven what happens at

a cellular level. Observing what occurs at a cellular level at different stages with the use of

different microcopy techniques can explain if there is any difference as in damage caused to the

cells by bruising. Bruise research has been done mainly with stored apples, which are physically

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older fruit. Older fruit contains more air spaces, due to the degradative changes that occur in the

cell. The degradation of large pectin molecules found in the middle lamella that bind together the

walls of neighboring cells (Kays, 1997). The dissolution of the middle lamella makes the tissue

more brittle and possibly with a different bruise mechanism than fresh apples. According to Pitt

(1982), Pitt and Chen (1983) Holt and Schoorl (1982, 1983b, 1984b), Van Woensel and De

Baerdemaeker (1983), Lin and Pitt (1986), Vincent (1990), Gao and Pitt (1991) Roudot et al.

(1991), Khan and Vincent (1990, 1991, 1993ab) Abbott and Lu (1996) Loodts et al. (2006)

bruising is a consequence of breakage of intercellular bonds, propagation of cell wall ruptures, or

cell deflation as a result of loss and diffusion of cell fluid. These conclusions resulted from

analyses at a cellular level, modeling cell mechanism response to external loads. With the use of

different microscopy techniques, it will be possible to understand what occurs in the damaged

tissue/area after being picked and bruised at different maturity levels.

The contributions of this work are, 1) identification of the numerous techniques used to

observe bruised tissue, 2) examination of freshly harvested apples at different ripening stages

without changes due to storage time or temperature, and 3) comparison of fresh vs. stored fruit

response to bruising.

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MATERIALS AND METHODS

EXPERIMENTAL DESIGN AND TREATMENTS

Apples for this study (Malus domestica Borkh cv. ‘Golden Delicious’) were harvested

from a commercial orchard located in Orondo, WA over two years (2006-2008). The experiment

was laid out as a completely randomized design. Fruit were harvested at different maturity levels

based on skin color. Green apples were harvested 143 days after full bloom (dafb) in 2007 and

132 dafb in 2008; creamy colored apples 150 dafb in 2007 and 139 dafb in 2008; and yellow

apples 158 dafb in 2007 and 146 dafb in 2008. The orchard was managed as a commercial

organic orchard. Irrigation, fertilizer and spraying practices were the same across all treatments.

FRUIT MATERIAL

Six fruit from three different trees was harvested at the three intervals during the

commercial harvest season, as detailed above, for a total of three treatments based on maturity

index/skin color. During harvest, fruit was selectively hand picked based on the maturity index

color change described by Mitcham et al. (2006) (the intermediate white color was added) and no

apparent damage. After harvest, the apples were transported in tri-layered European apple boxes,

nested in cylindrical holes cut in low-density foam with an additional foam sheet on the bottom

of the box to protect the apples from damage. In the lab, pickers’ bruises were simulated and the

treated fruit was left at room temperature for 48 hours. An Instron Model 1350 (Instron Industrial

Products, Grove City, PA) was used to simulate the external force applied by the pickers on the

apples using a silicon finger attached to the machine to create a finger print force sufficient to

bruise the apples. Apples were all bruised at the longitudinal location. Tissue samples were

observed using confocal and scanning electron microscopes 48 hours after bruising.

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FEI SEM TISSUE

Tissue from bruised apples was cut into approximately 1 cm thick sections and observed

using a Quanta 200F SEM (FEI Company, Hillsboro, Oregon) , under low vacuum mode.

Depending on the bruise size, the whole discolored area was cut in half and observed under the

microscope (fig. 1). Since the experimental methods for studying in situ deformation are limited

and that the tissue is not studied in its natural state due to the procedures used to preserve the

tissue, this method was selected to analyze the tissue under its natural state. With this technique,

we expected to see what really occurs (the natural state of the bruised tissue) in the tissue without

being altered by the fixing or dehydration procedures of the other techniques.

FLUORESCENCE MICROSCOPY

The LSM 510 meta laser scanning microscope (Carl Zeiss MicroImaging Inc,

Thornwood, New York,) was used to observed live and dead tissue using the fluorescent dyes

Calcofluor white (1 drop mixed with 1 drop of distilled water) and 5(6)-CFDA, SE; DFSE (5-

(and-)6-carboxyflourescein succinimidyl ester, mixed isomers (CDFA, C1157) from Invitrogen

Corporation (Carlsbad, CA). Four ml of DMSO was mixed with 25 mg of CFDA and 1 µl of this

solution mixed with 1 ml of distilled water. The bruise was cut and 0.5 mm thick slices of tissue

were mounted onto white snow coat micro slides (1” x 3” x 1.00 mm) and dyed with CDFA for

10 minutes. A drop of the calcofluor solution was added and covered with a microscope cover.

69

RESULTS

SEM

Using SEM, it was observed that bruise damage starts under the collenchyma cell layer,

approximately six layers under the epidermis. Where larger intercellular spaces were located,

there was a large amount of dead, burst, or crushed cells. More cells were damaged as harvest

dates progressed, and the damage was observed to be adjacent to the larger airspaces and

transmitted to the neighboring cells (fig. 2). In all treatments the hypodermis was intact. In

some cases, some compactness of the hypodermis layer was observed (fig. 3), however, there

was no apparent damage (fig. 4 - images of green stage under SEM).

Apple parenchyma tissue was observed to suffer varying mechanical failures depending

on the force applied. Contrary to what Upchurch (1986) found, the presence of larger

intercellular spaces in undamaged tissue, our findings show that tissue with larger airspaces is

more vulnerable to bruising. As Tu et al. (1996) observed, larger intercellular spaces are due to

degradation of the middle lamella and a reduction of cell adhesion or part of the apple’s tissue

properties. Zamorskyi (2007) observed that ‘Golden Delicious’ apples have denser intercellular

spaces and uneven cell size and structure which also confirms what Alvarez et al (2000b)

reported regarding inhomogeneities in structure that weakens tissue and makes it more

vulnerable to damage.

FLUORESCENCE MICROSCOPY

Use of CFDA and Calcofluor dyes indicated cells in the bruised area were a mixture of

dead and live cells. Cells adjacent to larger airspaces were dead and the amount of dead cells

varied depending on the maturity of the apple. The more mature the fruit was, the greater the

number of dead cells surrounding the airspaces. The hypodermis layer of cells was generally

70

intact, beneath which areas of dead cells were visible (fig. 4). These observations confirm those

of Esau (1965) who reported stronger cells in the top layers (epidermis of hypodermis) made up

of collenchyma cells and weaker cells in the lower deeper area (cortex), parenchyma cells. Using

confocal microscopy, Tu et al. (1996) observed larger intercellular spaces in mature fruit.

Intercellular spaces increase as apples ripen due to increases and decreases in contact among

cells (Vincent, 1989). Use of fluorescent dyes, enabled us to identify cell walls and intact cell

membranes in the hypodermis and, in other areas, intact cell wall structures with damaged cell

membranes. In burst or crushed cells the remains of the cell wall structure were easily identified

using the Calcofluor dye.

DISCUSSION

Numerous and varied definitions and symptoms of bruising can be found in the literature.

A bruise can be due to cell injury that results in flesh browning (Mohsenin et al. 1962); a

discoloration or fracture of the tissue (Chen et al.1986); cell bursting (Holt and Schoorl 1977);

cell rupture (Diehl et al.1979; Pitt 1982) and flesh browning (Robitaille and Janick 1973); an

injured area which is flat, soft and brown with a high respiration rate (Ericsson and Tahir 1996);

a dark spot (Blahovec 1999); or just an indentation of the surface and a slight discoloration

(Crisosto et al.1993) where tissue is compressed or impacted. Based on our observations, we

would define a bruise as an area of discolored tissue comprising an array of undamaged and burst

and crushed cells, along with cells that have not been physically damaged. The discoloration is

only an indication of where the damage can be found. This can occur inside the cell if the cell

vacuolar membrane is damaged or outside it the cell contents is released to the intercellular

spaces when the cells tenses.

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It appears that the collenchyma cells transmit the applied force to the underlying

parenchyma cells, sustaining no apparent damage themselves as Knee and Miller (2002)

reported. Similarly, we observed that collenchyma cells were not damaged, while the

parenchyma cells absorbed all the energy and therefore burst or were crushed, resulting in cell

death. All our observations are consistent with those of other investigators. Mohsenin (1970)

showed that in a bruised area, layers of cells remain unbroken, and even among the damaged

layers, groups of cells survived. Holt and Schoorl (1983) estimated that only 0.03 % of cells are

fractured in bruised areas. Using TEM, we did not observe cell membrane damage (data not

shown), however, such damage has been reported by Rodriguez et al (1990).

At an angle of 45° relative to the direction of the applied load, we observed a crack (large

intercellular space), in addition to a crack in a parallel plane to the applied load within the fruit

tissue (fig. 5). This could be due to the morphology of ‘Golden Delicious’ apple tissue.

Zamorskyi (2007) observed that ‘Golden Delicious’ apples have denser intercellular spaces and

uneven cell size and structure. Alvarez et al. (2000b) noted irregularities in tissue structure

weakens the tissue and makes it more vulnerable to damage.

The crack parallel to the load was not surrounded by discolored tissue. This may be due

to minor damage in the tissue such as Holt and Schoorl (1983) observed in potato tissue (fig. 6).

In our research, cell bursting and detachment were observed under SEM (fig. 2) but this was not

observed in detailed under fluorescence microscopy (data not shown). As Donald (2003)

explained, experimental methodologies for studying in situ deformation are limited and it is

uncertain whether the procedures used to preserve the tissue, such as vacuum for dehydration

and coating in SEM, fixing and embedding for light microscopy alter tissue structure.

Harker et al. (1997b) showed that cell separation without rupture may be common in fruits that

soften when ripe, such as peach, kiwi and strawberry. In pears, cellular failure in unripe tissue

72

was due to cell wall failure and cell fracture, but in ripe tissue, failure was due to intercellular

debonding (De Belie et al. 2000). The main component of intercellular adhesion is the extent of

intercellular contact, which is determined by cell shape and packing, water loss and size or

absence of intercellular spaces. These factors change as fruit ripens, leading to larger air spaces

and reduced intercellular contact (Glenn and Poovaiah 1990; Hallett et al. 1992; Harker and

Sutherland 1993), allowing increased tissue deformation under stress. Pierzynowska-Korniak et

al. (2002) showed that different apple cultivars have different cells shapes which contribute to

their unique mechanical properties.

We observed greater damage with increasing apple maturity due to larger airspaces and

cell detachment, causing a larger area of discolored, damaged tissue. This confirms observations

by Vincent et al. (1991) that airspaces are the weakest part of the tissue.

CONCLUSIONS

As fruit matures and ripens, the dissolution of the middle lamella of the cell wall results

in softening of the tissue. During senescence, there is a progressive loss of membrane integrity,

as well as a loss of cell compartmentalization. In stored apples, there is more airspace between

cells due to the detachment of the cells. For these reasons it is more difficult to understand

bruising mechanisms in stored or senescing tissue.

Our major findings regarding the mechanism of bruising of freshly harvested ‘Golden

Delicious” is based on apple tissue properties (figs. 7 and 8): 1) cell wall rupture or damage is

not required for bruise induction; 2) parenchyma cells are involved in the discoloration of

bruised tissue in ‘Golden Delicious’ apples because they are more fragile than the collenchyma

cells; 3) under tissue compression, multiple failure modes might be involved in parenchymatic

tissue damage; 4) cracking as well as rupture might be present in the damaged tissue; 5)

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damaged tissue is made up of a mixture of dead and live cells; 6) cell rupture is facilitated by

intercellular spaces which are the weakest point in the tissue; 7) cells adjacent to intercellular

spaces are more vulnerable to damage due to the lack of cell-to-cell contact, thereby allowing

them to expand and rupture more easily than cells that are tightly packed; and 8) discoloration of

bruised tissue might be conferred by damaged cells that have released their contents to

neighboring tissue. This might help explain why discoloration often outlines the ‘crack’ in the

bruised tissue.

ACKNOWLEDGEMENTS

The authors recognize the support of the Agricultural Research Center, Washington State

University and the Consejo Nacional de Ciencia y Technologia, Government of Mexico.

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Acta Horticulturae 746:509-512.

77

Figure 1. Discolored (bruised) area of compressed “Golden Delicious’ apple.

78

Air space

Healthy cells beside air space

a

Air space area

Increased Damaged cells beside air space

Dead cells

Live cells

b

Figure 2. FEI SEM images of control and bruised tissue. a) control tissue, b) bruised tissue.

79

b a

Figure 3. Control and bruised tissue. Compressed area of the hypodermis in bruised tissue. a) Control tissue, b) Bruised tissue.

80

c

a b

Figure 4. Confocal imaging of bruised and undamaged apple tissue. a) Damaged cells (parenchyma cells) under undamaged hypodermis cells (collenchymas cells)(blue dye for dead cells and green dye shows live cell membranes), b) Dead cells in bruised tissue, c) Control apple tissue. Healthy undamaged cells.

81

Figure 5. A 45° crack from the applied force.

82

Figure 7. Proposed bruising mechanism for ‘Golden Delicious’ apples.

b a

Figure 6. Crack parallel to the load a) fresh bruised apple, b) ESEM image, bruised tissue (gold coated).

83

White bruised

crushed cells

burst cells

intact cells

Figure 8. Confocal image of cell wall and membrane damaged due to bruising.

Figure 9. Difference in intercellular spaces of fresh and stored apples. a) Fresh unbruised tissue, b) Stored unbruised tissue.

84

CHAPTER THREE

Harvesting by Peel Color to Reduce Bruising of “Golden Delicious’ Apples

K. Mitsuhashi1, C. D. Clary2*, J. K. Fellman3, M. J. Pitts4 and E. A. Curry5

1Graduate Student, 2Assistant Professor, 3Professor, Department of Horticulture and Landscape

Architecture, Washington State University, Pullman, Washington; 3Associate Professor,

Biological Systems Engineering, Washington State University, Pullman, Washington; 5 Research

Plant Physiologist, U. S. Department of Agriculture, Agriculture Research Service, Tree Fruit

Research Laboratory, Wenatchee, Washington.

*Corresponding author: Carter D. Clary, Department of Horticulture and Landscape

Architecture, P.O. Box 646120, Washington State University, Pullman, WA, 99164-6414,

phone: 509-335-6647; fax: 509-335-8690; email: @wsu.edu.

Abstract

‘Golden Delicious’ apples harvested at three peel color stages were immediately bruised

to a constant depth using an artificial silicon finger attached to an Instron universal material

testing instrument. Bruised tissue was sliced sequentially from the fruit surface in a plane

perpendicular to the direction of the applied force until discoloration was no longer evident.

Thickness and discolored area of each tissue slice was measured using a digital caliper and total

volume of bruised tissue was estimated. Analysis showed there was a significant difference in

bruise volume between green and yellow peel stages. Susceptibility to bruising of fruit at the

white (intermediary) stage appeared to vary with environmental condition (year to year).

Compared with the previous year. The 2008 growing season was cooler and shorter and bruise

85

volume at all stages was greater. Analysis of fruit maturity suggested bruise volume was

influenced by ripening-induced changes in cell structure, size and integrity. Where fruit peel

color changed but flesh firmness (Magness-Taylor) did not, bruise volume increased. We

conclude that peel color is a better indicator of bruise susceptibility than fruit flesh firmness.

Introduction

‘Golden Delicious’ apples are usually harvested in a single pick depending on peel

ground color, compared to other cultivars such as ‘Gala’ that are harvested more than once based

on both development of red pigmentation and changing ground color. ‘Golden Delicious’ apples

are harvested approximately 130 days after full bloom (DAFB) (Jackson, 2003) at which time,

depending on the season, peel color may vary from green (less mature) to yellow (more mature).

Roth et al. (2005) reported firmer apples bruise more than softer apples. Hertog et al.

(2003) found that firmness measured by a Magness-Taylor fruit penetrometer (Stable Micro

Systems Ltd., Godalming, Surrey, UK) is affected by the mechanical strength of the tissue.

Others have reported apple fruit flesh firmness is determined by the physical anatomy of the

tissue including such components as cell size, cell shape and tissue arrangement (Toivonen and

Brummell, 2008). Moreover, cell wall thickness and strength, cell to cell adhesion, and turgor

may influence bruise volume (Harker et al. 1997). Further, development of internal fruit

structure is influenced by seasonal temperatures which may alter the number of cells in the

cortex (Lakso et al. 1995). Small cells tend to have greater cell to cell contact, a lower amount of

intercellular air space, and less cell sap (cytoplasm and vacuole), making the tissue firmer and

less juicy (Harker et al. 1997).

86

During ripening, fruit flesh undergoes degradation resulting in reduced cell wall thickness

and firmness and lessened intercellular adhesion (Toivonen and Brummell, 2008). Fruit

parenchyma cells often have relatively thin and weak primary cell walls compared to those from

more vegetative (structural) tissues, which have a higher proportion of cells with thickened and

lignified cell walls, thereby conferring greater strength (Kays, 1997; Toivonen and Brummell,

2008). Roth et al. (2005) also reported that softer fruits are less sensitive to damage by impact

than firmer fruit and therefore bruise less. They showed that reduction in firmness of ripened

fruit was attributable, in part, to increased enzymatic activity leading to cell wall breakdown.

Specifically, polygalacturonase activity increased exponentially during the first 30 days after

harvest.

Riper apple tissue has more intercellular spaces and is not as dense as younger, immature

tissue (Glenn and Poovaiah, 1987; Roth et al. 2005). According to Alvarez et al. (2000ab) the

closer the cells are to each other, the smaller the intercellular airspaces are and the less damage

occurs when a force is applied. Indeed, Knee (1993) has suggested an increase in the number

and size of individual air spaces during ripening may partially account for the decline in

firmness.

Previous research on bruising of ‘Golden Delicious’ focused on comparing peel color

stages green vs. yellow for bruising susceptibility. Hyde and Ingle (1968), Kvaale et al. (1968),

Diener et al. (1979) and Saltveit (1984) all concluded, to reduce bruising of ‘Golden Delicious’

apples at harvest, it is advantageous to pick fruit while the peel is green.

Preliminary observations of bruising at harvest within several commercial ‘Golden

Delicious’ orchards in central Washington (Mitsuhashi, unpublished) indicated a transitory

‘white’ peel color may also be related to bruise susceptibility. The objective of this research was

87

to determine if bruising of ‘Golden Delicious’ apples at harvest could be reduced by evaluating

differences in bruise susceptibility (volume) of fruit harvested at the white peel ground color

compared with that of fruit harvested with green or yellow peel color.

Material and Methods

Experimental design and treatments

This study was completed over two harvests (2007-2008) in a commercial apple (Malus

domestica Borkh cv. ‘Golden Delicious’) orchard located in Orondo, WA. The experiment was

laid out as a completely randomized design with three treatments (peel color) each consisting of

3 fruit harvested from each of 20 trees. Sixty green, white and yellow apples were harvested at

143, 150 and 158 DAFB, respectively, in 2007 and 132, 139 and 146 DAFB, respectively, in

2008, as described by Mitcham et al. (2007). The orchard was managed organically and all trees

were treated similarly.

Bruise evaluation and fruit quality analysis

Apples were harvested individually into tri-layered European-style (Euro) apple boxes

and nested in cylindrical holes cut in low-density foam. An additional foam sheet lined the

bottom of the box to protect the apples from damage due to handling after harvest which was not

the focus of this work. Fruit were transported to the laboratory and left at 22 °C for 48 hours to

establish temperature equilibrium.

Before bruising, each fruit was weighed. To simulate bruising by a picker, a silicon

finger was attached to an Instron Model 1350 (Instron Industrial Products, Grove City, PA) and

displaced 10 mm in 23 seconds into the side of the apple at approximately the widest equatorial

diameter (shoulder). Rate of displacement of the silicon finger was the same for all apples. Fruit

88

were then placed back into the Euro foam boxes and kept at 22 °C and 68% relative humidity for

48 hours prior to evaluation.

Bruise volume was measured by cutting successive 1mm tangential slices through each

bruise, perpendicular to the direction of the applied force, using a stainless steel blade, until

discoloration (brown) was no longer evident. Bruised (brown) region of each slice was

measured at the widest diameter and again at 90° to the widest diameter using a digital caliper

(Model CD67-6, Mitutoyo Corporation, Kawasaki, JP) and averaged to provide an average

diameter of a circular (assumed) area. Bruise volume per tissue slice was estimated to be the

area x thickness (depth). Total bruise depth was calculated as the sum of the depths of affected

slices. Total bruise volume was calculated in cubic millimeters using the sum of bruise volumes

of individual slices.

Apple quality evaluation

After fruit were bruised, but before slicing for bruise volume determinations, flesh

firmness was measured using a texture analyzer (FTA, Güss Manufacturing (Pty) Ltd., Strand,

SA) on opposite sides of each apple after removing about 1 mm peel tissue.

After fruit were processed for bruise volume evaluation, soluble solids content (SSC) was

measured from the juice of the apple using an ABBE Refractometer (AO American Optical,

Model 10450 Mark II, Digital Refractometer) and recorded as % Brix. To measure starch

clearing index, each fruit was cut transversely through the equatorial line and the cut surfaces

dipped in 2 % iodine/potassium iodide solution (1:4) for 10 seconds and left to equilibrate for

about 1 minute. The stained area of each fruit was compared to a standard scale of 1 to 6 (1 =

maximum starch content, 6 = no starch content) and the values recorded.

89

Acid content expressed in percent malic acid equivalents was measured by a titration

method using the Model TIM 850 titration workstation (Radiometer Analytical SAS, Lyon

France).

Peel ground color was measured using a Konica Minolta Croma Meter CR 300 (Konica

Minolta Sensing, Inc., Tokyo, Japan). Values were expressed in CIE color system with the

L*a*b axis representing lightness, green-red and blue-yellow respectively and transformed to

degrees (°) hue according to Mclellan et al., 1995.

Internal ethylene concentration (IEC) in each fruit was evaluated by first inserting an 18-

gauge needle equipped with a rubber septum through the fruit calyx into the central cavity and

withdrawing 1mL of core gas. Ethylene in the withdrawn gas sample was measured by injecting

0.5mL of the gas into an HP 5880A gas chromatograph with flame ionization detector (GC-FID,

Hewlett Packard, Avondale, PA, USA) equipped with a 1m × 3.2mm i.d. glass column packed

with 80–100 mesh Porapak Q (Waters Corporation, Milford, MA) in an oven at 40 ◦C according

to standard methods.

Statistical Analysis

Data collected from the two-year trial were combined and analyzed for statistical

significance using the general linear model (GLM) procedure of SAS v.9.1 software (SAS

Institute, Cary, NC). Differences among treatments were identified using the Duncan’s New

Multiple Range Test at P < 0.05. Data within variables were distributed normally.

90

Results

Fruit Quality 2007 vs. 2008

In 2008, anthesis was delayed by about 2 weeks due to cooler than normal ambient

temperature (data not shown). Because fruit were harvested at about the same calendar date as

the previous year, however, the 2008 growing season was about 10-12 days shorter. Although

peel color at the time of sampling was similar in the two years (Table 1.) comparison of flesh

firmness, starch index, soluble solids and titratable acidity illustrate effect of seasonal

temperature differences on fruit quality.

In 2007, fruit flesh firmness in apples at green and yellow peel color stages was less than

that of fruit sampled at the white stage, whereas in 2008, firmness of green apples was

statistically different than that of white and yellow apples (Table 1). In 2007 and 2008, firmness

was higher in white apples compared to green and yellow apples.

Starch content differed significantly among the three color stages and decreased with

successive harvests in both years (Table1). IEC also increased with each subsequent sampling,

however, only the yellow peel stage showed IEC greater than 1.0 μl·l-1. In 2007, although green

apples had the lowest titratable acidity, there was no significant difference between white and

yellow apples showed. In 2008, green and yellow apples showed no significant difference, yet

both differed significantly from white apples. Soluble solids content in both 2007 and 2008

show all stages were significantly different, although in 2008 there was an unexplained increase

during the white peel stage (Table 1). Also in 2008, titratable acidity in white apples was lower

than that in green and yellow apples; though not common, this pattern is not consistent with

normal progressive ripening. Although peel color was correlated with starch content, we did not

91

find significant correlations among starch, titratable acidity and soluble solids content in either

year (data not shown).

Bruise volume

The relationship between peel color and bruise volume was similar for both years

showing a general trend toward greater bruise volume with increasing fruit maturity (Fig. 1). In

contrast, the degree to which bruise volume occurred at both green and white peel stages was

significantly different from 2007 to 2008. Bruise volume at green and white stages between

years was significantly different. Bruise volume of apples harvested at the white color stage was

similar to the green stage in 2007 and the yellow stage in 2008. In other words, apples at the

white peel stage had a tendency to behave as green apples one year and yellow apples the next.

Thus, white peel color stage may not be useful for predicting bruise volume unless the specific

environmental and fruit quality parameters are being evaluated as well.

Discussion

Using starch index and IEC as criteria, yellow apples in 2007 and 2008 were at similar

stages of physiological maturity and showed similar values for bruise volume, even though fruit

firmness differed by more than 2 lbs (Table 1, Fig. 1). This suggests that fruit firmness is not

correlated with bruising. Indeed, Pearson Correlation Coefficients for bruise volume vs. flesh

firmness for 2007 and 2008 are quite low (Fig. 2). Further analysis indicates that at none of the

peel color stages was fruit firmness correlated with imposed bruise volume (Table 2).

These data might also signify that firmness as measured by the Texture Analyzer is not

the proper instrument to predict cell to cell attachment and bruise susceptibility. Other

investigators (Lidster and Tung, 1980; Klein, 1987; Ericson and Tahir, 1996) found no

92

relationship between fruit characteristics such as firmness or density and bruise susceptibility. In

their study, there was no apparent relationship between bruising, color or firmness.

Ericson and Tahir (1996) bruised apples by dropping them from a certain height, and

determined that smaller fruit was less prone to bruising due to high impact energy during

dropping than was larger fruit. They did not find difference in harvest dates. Viljoen et al.

(1996) and Ericson and Tahir (1996) picked based on dafb and found no significant relationship

between bruise volume and color. In the present study, apples were of approximately the same

weight, but still exhibited different levels of bruising.

In fruit such as apple, softening involves degradation of large pectin molecules in the middle

lamella that bind cells walls together (Kays, 1997). During ripening, enzymatic degradation of

cell walls results in a textural alteration (Kays, 1997). Fruit cell firmness decreases during

storage while soluble pectin increases and detachment of cells occurs (Glenn and Poovaiah,

1987). The number of damaged cells also increases with ripening, as does the size of

intercellular spaces (Figure 5).

In the present study there was a significant difference in bruise volume using color as an

indicator. Among years, bruise volume was markedly different, especially at the green and white

peel stages. This might have been due to seasonal differences in tissue characteristics since cell

division and number influence fruit tissue characteristics. ‘Golden Delicious’ apples show a

positive curvilinear growth curve in the early part of the season from 32 to 74 days and cultivar

and environment contribute to the number of cells in the cortex (Lakso et al. 1995). Smith

(1940, 1950) found that cultivar differences in apple size were due to differences in cell division

(cell number) and cell size. This could help explain why some apple varieties are more bruise

resistant than others. In many traditional apple and pear fruit areas, fruit tends to be smaller

93

during cooler seasons or when grown under cool conditions by reason of latitude or altitude.

Perhaps, by developing models of fruit growth using multiple environmental variables, we might

be able to predict tissue properties and bruising sensitivity of apples.

Conclusions

These data suggest severity of picker bruising in ‘Golden Delicious’ might be reduced by

picking apples while they are green. Nevertheless, although green apples bruised less than white

and yellow apples at constant depth (as imposed by the silicon finger), they are also more firmly

attached to the tree and one must exert a greater force to remove them. Thus, proper technique is

key to minimizing picker-induced bruising.

94

References

Alvarez, M. D., Saunders, D.E.J., and J.F.V. Vincent. 2000a. An engineering method to evaluate the crisp texture of fruit and vegetables. Journal of Texture Studies, 31:457-473.

———. 2000b. Fracture properties of stored fresh and osmotically manipulated apple tissue. Eur Food Res Tech, 211:284-290.

Diener, R. G., Elliot, K. C., Nesselroad, P. E., Ingle, M., Adams, R. E., and S. H. Blizzard. 1979. Bruise energy of peaches and apples. Transactions of the ASAE, 22:287-290.

Ericsson, N. A., and I.I. Tahir. 1996. Studies on apple bruising. II. The effects of fruit characteristics, harvest date and precooling on bruise susceptibility of three apple cultivars. Acta Agric. Scand. Sec. B. Soil and Plant Sci., 46:214-217.

Glenn, G. M., and B.W. Poovaiah. 1987. Role of calcium in delaying softening of apples and cherries. Postharvest Pomology Newsletter 5 (1):10-19.

Harker, F. R., R.J. Redgwell, I.C. Hallett, S.H. Murray, and G. Carter. 1997. Texture of Fresh Fruit. Hort. Rev. 20:121-124.

Hertog, M. L. A. T. M., Ben-Arie, R., Roth, E., and B.M. Nicolai. 2003. The effect of humidity and temperature on invasive and non-invasive firmness measure. Postharvest Biol. Technol. 33 (1):79-91.

Hyde, J. F., and M. Ingle. 1968. Size of apple bruises as affected by cultivar, maturity and time in storage. Proc. Amer. Soc. Hort. Sci. 92:733-738.

Jackson, J. E. 2003. Biology of horticultural crops. Cambridge: Cambridge University Press. Kays, S. T. 1997. Postharvest physiology of perishable plant products. Athens, GA: Exon press. Klein, J. D. 1987. Relationship of harvest date, storage conditions, and fruit characteristics to

bruise susceptibility of apple. journal of american society for horticultural science 112:113-118.

Knee, M. 1993. Pome fruits. In Biochemistry of fruit ripening, edited by T. G. Seymour. London: Chapman and Hall, 325-346.

Kvaale, A., Patterson, M.E., and R.B. Tukey. 1968. Lack of bruise recovery in golden delicious apples after storage. Paper read at Proc. Wash. State Hort. Ass.

Lakso, A. N., L. Corelli-Grappadelli, J. Barnard, and M.C. Goffinet. 1995. An expolinear model of the growth pattern of the apple fruit. Journal of horticultural science 70:389-394.

Lidster, P. D., and M.A. Tung. 1980. Effects of fruit temperature at time of impact damage and subsequent storage temperature and duration on the development of surface disorders in sweet cherries. Can. J. Plant Sci. 60:555-559.

Mclellan, M. R., Lind, L. R. and Kime, R.W. 1995. Hue angle determinations and statistical analysis for multiquadrant hunter L,a,b data. Journal of Food Quality 18:235-240.

Mitcham, E. J., C.H. Crisosto and A.A. Kader. 2007. Apple: 'Golden delicious'. UC Davis, 05/10/07 2007 [cited 01/03 2008].

Mitsuhashi, K.M., Unpublished. Roth, E., E. Kovacs, M.L.A.T.M. Hertog, E. Vanstreels and B. Nicolai. 2005. Relationship

between physical and biochemical parameters in apple softening. Acta Horticulturae 682:573-578.

Saltveit, M. E. 1984. Effects of temperature on firmness and bruising of 'Starkrimson' and 'Golden Delicious' apples. HortScience 19 (4):550-551.

Smith, W. H. 1940. The histological structure of the flesh of the apple in relation to growth and senescence. Jour. Pomol. and Hort. Sci. 18:249-260.

95

———. 1950. Cell-multiplication and cell-enlargement in the development of the flesh of the apple fruit. Ann. Bot. 14:23-38.

Toivonen, P. M. A., and D.A. Brummell. 2008. Biochemical bases of appearence and texture changes in fresh-cut fruit and vegetables. Postharvest biology and technology 48:1-14.

Viljoen, M., Mostert, I., Wepener,C., and H.M. Griessel. 1996. The effect of harvest time on the bruisability of golden delicious apples. Decidious fruit grower 46 (2):56-58.

96

Table 1. Fruit quality parameters for ‘Golden Delicious’ apples harvested at 3 peel color stages in 2007 and 2008.

Year

Harvest

(DAFB)z

Peel color

(visual)

Peel color

(° hue)

Weight

(g)

Flesh

firmness

(lb)

Starch

index

(1-6)

Soluble

solids

(°Brix)

Titratible

acidity (%

malic acid)

Internal

ethylene

(μl·l-1)

2007 143 Green 120.4

cy

225.1 b 17.13 a 3.23 a 11.76 a 0.526 a 0.28 a

150 White 111.1 b 203.1 a 18.17 b 3.54 b 13.54 b 0.559 b 0.74 a

158 Yellow 106.8 a 236.0 b 17.01 a 4.62 c 14.96 c 0.550 b 7.47 b

2008 132 Green 120.0 c 236.8 a 15.33 b 3.49 a 11.28 a 0.490 b 0.02 a

139 White 110.7 b 251.8 b 15.51 c 4.47 b 13.34 c 0.407 a 0.12 a

146 Yellow 103.7 a 250.3 b 14.99 a 4.97 c 12.71 b 0.463 b 5.74 b

zDAFB = Days after full bloom. yColumn values within a year followed by the same letter are not significantly different as determined by Duncan's New Multiple Range test (P < 0.05).

0

10000

20000

30000

40000

1 2 3 1 2 3

2007 2008

G W Y G W Y

Peel visual color

Bruis

e volu

me (m

m3 )

Figure 1. Bruise volume (imposed) at harvest as a function of year and peel color: green (G), white (W) and yellow (Y). Bars indicate + standard error of the mean.

97

12 14 16 18 20 22FIRM

0

20000

40000

60000

80000

100000

VOL

20082007

Bru

ise

volu

me

(mm

3 )

Fruit flesh firmness (lb)

r = - 0.27 r = - 0.18

Figure 2. Linear regression and Pearson correlation coefficient (r) for fruit flesh firmness vs. imposed bruise volume for ‘Golden Delicious’ in 2007(_________) and 2008 (---------).

98

Table 2. Pearson correlation coefficients for fruit flesh firmness vs. imposed bruise volume at 3 peel color maturity stages for ‘Golden Delicious’ in 2007 and 2008.

Year

Harvest

(DAFB)z

Peel color

(visual)

Correlation Coefficient

(r)

2007 143 Green -0.141

150 White 0.012

158 Yellow -0.445

2008 132 Green -0.064

139 White -0.192

146 Yellow -0.214

99

A

B

C

s s

s

s

s

s

s

s

s

s

s

s

s

s

s

s s

s

Figure 3. Grayscale light micrographs of ‘Golden Delicious’ apple peel cross section at progressive harvests in 2007: green (A), white (B) and yellow (C) peel color. Note thickening parenchyma differences in size of intercellular airspace (s) as apples ripen. Individual image view field = 1.0 mm (bars represent 500 µm).

100

SUMMARY

This Ph.D. research project proposes the idea that bruising damage is dependent upon cell type.

Because collenchyma cells are smaller, stronger and with less intercellular spaces than

parenchyma cells, the distribution of these cells in the tissue explains the damage caused by

bruising. The distribution of cells in apple is: six layers of collenchymas cells followed by the

cortex composed of parenchyma cells, thus damage starts where the first layers of parenchyma

cells are present. Bruised/discolored tissue is composed of dead and live cells. The fact that

tissue is discolored, doesn’t mean all cells that compose it are damaged.

Tissue with larger intercellular spaces suffers more damage due to the additional stress applied to

the cells that are closer to the large intercellular spaces. Bruise volume was affected by

intercellular spaces present in the damaged tissue.

As fruit ripens bruise volume increased due to cell decompartmentalization, which affects cell

wall strength and increases intercellular spaces and cell-to-cell detachment. Greener fruit is more

tightly pack and therefore suffers less damage then cells that are loosely attached as it occurs in

riper fruit (yellow peel color). Cell-to-cell contact will depend on peel color stages, as fruit

ripens there are more intercellular spaces and there will be more bruise volume.

Out of all the different techniques used in this project to identify bruising damage (SEM, ESEM,

light microscopy, TEM and Confocal microscopy) I would recommend the use of Confocal and

ESEM techniques for future research. These techniques proved simple and accurate. There is no

need to alter the composition or state by dehydrating the sample, there is no need to fix samples

and results can be obtained just after bruising.