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The Effect of Temperature, Oxygen Concentration
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presented by

Jose Luiz MOreira Garcia

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THE EFFECT OF TEMPERATURE, OXYGEN CONCENTRATION AND
STORAGE INTERRUPTION ON PHYSIOLOGICAL DISORDERS OF
'MCINTOSH' APPLES

By

Jose Luiz Moreira Garcia

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1980

ABSTRACT
THE EFFECT OF TEMPERATURE, OXYGEN CONCENTRATION AND

STORAGE INTERRUPTION ON PHYSIOLOGICAL DISORDERS OF
'MCINTOSH' APPLES

By

Jose Luiz Moreira Garcia

'McIntosh' apples are subjected to a physiological dis-
order termed 'brown core' when stored at 32°F, particularly
in controlled atmosphere (CA) low in 02 and high in 002'
Accumulation of toxic metabolites is thought to be a con-
tributing factor. Experiments were conducted with 'McIntosh'
to determine the effects of hypobaric storage at 32°F and CA
storage at 32° and 36°F in 1.5 and 3% 02 on the development
of physiological disorders. Storage treatments were applied
continuously or intermittently by returning fruits to an
atmosphere of air at the storage temperature or to 68°F.

Brown core was prevalent in fruits stored at 32°F and
in 3% 02 and was not attenuated by storage interruption. It
worsened during 7 days at 68°F after 7 months of storage.

Low temperature breakdown was observed in only one instance
and was not related to storage temperature, 02 level or
storage interruption. The incidence of senescent breakdown
increased as the 02 level increased and was greater in fruits
stored at 36° than at 32°F. The same results were found for
scald with respect to 02 level.

CA and hypobaric storage retarded the loss of acidity

in.'McIntosh'. Concentration changes in organic acids of

Jose Luiz Moreira Garcia

'Empire' apples were the same during aerobic and anaerobic
metabolism at 68°F; malic decreased, citric remained con-

stant, and succinic and fumaric acids increased.

ACKNOWLEDGMENTS

The advice and encouragement of Dr. David R. Dilley is
gratefully acknowledged. I also thank Dr. Robert C. Herner.
Dr. Donald H. Dewey and Dr. Hugh C. Price for their help and
useful suggestions.

Financial assistance from a Consortium for the Develop-
ment of Technology - CODOT scholarship in the first year of
study and lately from a Conselho de Desenvolvimento Cienti—
fico e Tecnologico — CNPq scholarship is thankfully recog—

nized.

ii

TABLE OF CONTENTS

Page

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . vi

LI ST OF FIGURES I I I I I I I I I I I I I I I I I I I Vii

INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1

REVIEW OF LITERATURE I I I I I I I I I I I I I I 3

Brown Core of Apples . . . . . . . . . . . . . . 3

Causal Factors . . . . . . . . . . . . . . . . 3

Effects of Pre-harvest Factors . . . . . . . 4

Effects of Pre-storage Factors . . . . . . . 5

Effects of Fruit Factors . . . . . . . . . . 6

Effects of Storage Factors . . . . . . . . . 7

Effects of Post-storage Factors . . . . . . . ll

Association of Brown Core to Other Disorders . . 12

Low Temperature Breakdown . . . . . . . . l3
Alleviation of Brown Core and Low Temperature

Breakdown I I I I I I I I I I I I I I I I I I I I I la

Hypothesis Advanced to Explain Brown Core and Low

Temperature Breakdown . . . . . . . . . . . . . . . 16

Brown core I I I I I I I I I I I I I I I I I I I 16

Low Temperature Breakdown . . . . . . . . . . . 17

Organic Acid Metabolism . . . . . . . . . . . . . . l9
Behavior of Apple Fruits Under Low Oxygen and

Anaerobic Conditions . . . . . . . . . . . . . . . 24

MATRIALS AND METHODS I I I I I I I I I I I I I I I I 27

Fruit Material I I I I I I I I I I I I I I I I I I 27

Treaments I I I I I I I I I I I I I I I I I I I I 28

iii

First Experiment .
Second Experiment

Third Experiment

Fruit Maturity at Harvest

Fruit Firmness . .

Internal and External Disorders

Brown Core

Low Temperature Breakdown
Senescent Breakdown

Scald . . . . .

Titratable Acidity
Organic Acids . . .

Organic Acids Extraction .

Organic Acids Analysis .

Carbon Dioxide Determinations

Statistical Analysis
RESIJLTS I I I I I I I

First Experiment
Second Experiment .
Third Experiment .

Organic Acids .
Malic Acid . . .
Citric Acid .

Succinic Acid .
Fumaric Acid . .

Carbon Dioxide Production

DISCUSSION . . . . . .

First Experiment .
Second Experiment .
Third Experiment

iv

Page

28
29
33
34
3a
34

35
35
36
36

36
37

37
38
no
40

42

1+2
as

55

55
55
57

57
60

62
6h

65
69
73

Organic Acids Extraction and
Organic Acids

Malic Acid
Citric Acid
Succinic and Fumaric Acid
Carbon Dioxide Production

SUMMARY . . . .
LITERATURE CITED

Page

Analysis . . . . . 73
. . . . . . . . . 75

. . . 75

. . . . . . . 75
. . 76

. . . . . . . . . . 76
. . . . . . . . . 79

. . . . . . . . 81

Table 1.

Table 2.

Table 3.

Table A.

LIST OF TABLES

Firmness and titratable acidity of 'McIntosh'
apples stored for 7 months under different
temperature, atmosphere composition and
storage interruption regimes . . . . . . . .

Physiological disorders of 'McIntosh'

apples stored for 7 months under different
temperature, atmosphere composition and
storage interruption regimes . . . . . . . .

Effect of temperature, oxygen concentration,
atmospheric pressure and storage interrup—
tion on firmness and titratable acidity of
'McIntosh' apples . . . . . . . . . . . . .

Effect of temperature, oxygen concentration,
atmospheric pressure and storage interrup-
tion on physiological disorders of 'McIntosh'
apples after 7 months in storage and after
7 days at 2000 I I I I I I I I I I I I I I I

vi

Page

43

45

48

52

Figure

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

Schematic representation of the hypobaric
storage system used in the second
experiment . . . . . . . . . . . . . . . .

Separation of the tricarboxylic acid cycle
acids by HPLC . . . . . . . . . . . . . .

Changes in malic acid content of 'Empire'
apples kept in air or nitrogen atmospheres
at 2000. Each data point represents an
average of 3 determinations using indivi-
dual fruits. Standard deviations of the
means are shown for each data point . . .

Changes in citric acid content of 'Empire'
apples kept in air or nitrogen atmospheres
at 200C. Each data point represents an
average of 3 determinations using indivi-
dual fruits. Standard deviations of the
means are shown for each data point . . .

Changes in succinic acid content of 'Empire'

apples kept in air or nitrogen atmospheres
at 2000. Each data point represents an
average of 3 determinations using indivi-
dual fruits. Standard deviations of the
means are shown for each data point . . .

Changes in fumaric acid content of 'Empire'

apples kept in air or nitrogen atmospheres
at 20°C. Each data point represents an
average of 3 determinations using indivi-
dual fruits. Standard deviations of the
means are shown for each data point . . .

002 evolution of 'Empire' apples kept in

air or nitrogen at 20°C. Each data point
represents an average of 8 determinations
using individual fruits . . . . . . . . .

vii

Page

31

39

56

58

59

61

63

INTRODUCTION

Physiological disorders are often the primary factor
limiting the preservation period of fresh apples during con—
ventional refrigerated or controlled atmosphere storage. The
disorders usually appear toward the end of the cold storage
period and some become worse after transferring the fruits
to warmer temperatures during the marketing period. This
increases the magnitude of the problem. 'McIntosh' is par-
ticularly susceptible to the brown core disorder which has
been responsible for considerable losses of this cultivar
during storage.

The causal factors for several of the physiological dis-
orders are unknown but lately the accumulation of some meta—
bolites, namely tricarboxylic acid (TCA) cycle acids, had
been implicated as a factor responsible for the appearance
of some disorders. When certain of these metabolites accu-
mulate, they are toxic and may eventually kill the tissue
which in turn discolors. The incidence of some physiological
disorders, especially brown core and low temperature break-
down, can be lessened in some cultivars by briefly returning
the fruits to warm air at intervals during the storage
period. This interruption treatment is thought to dissipate
the accumulated metabolites allowing the fruits to be stored

for a longer period of time.

.1

This study was conducted to investigate the incidence
of physiological disorders as influenced by temperature and
oxygen concentration during storage. Also investigated was
the effect of interim warming treatment and aeration at low
temperatures at the midpoint during the storage period.

Analysis of TCA cycle acids was made to determine if
oxygen deprivation causes changes in these metabolites that

may lead to the development of physiological disorders.

REVIEW OF LITERATURE

This review of literature will deal primarily with
physiological disorders of the 'McIntosh' cultivar with
special reference to brown core since this disorder is the
main problem encountered during the storage of this cultivar.
Some attention will also be given to low temperature break-
down and to attempts to control both disorders in stored
apples. The physiological and biochemical considerations
pertinent to the subject will also be reviewed.

The nomenclature adopted for internal physiological
disorders of apples is the one suggested by Smock (107)
which is an agreement made by investigators from around the
world concerning the terminology for those disorders.

A general description of the symptoms of the disorders
dealt with in this review can be found in several other re-

views (26, 28, 81, 85, 125).
Brown.Core of Agples

l. Causal Factors

There is a great deal of controversy in the literature
concerning the causal factors of brown core of stored apples.
but the causes most frequently reported are: low storage

temperature, high carbon dioxide levels and senescence.

However, most workers seem to agree that 'McIntosh' is the
most susceptible variety to this disorder.
The factors that may influence the appearance of brown
core are listed below.
a. Effects of Pre-harvest Factors
(1) Growing season
Susceptibility to brown core seems to vary from
year to year with different climatic conditions during the
growing season (13, 28, 106, 109, 110), and there are some
indications that it may be worse following growing seasons
that have been cloudy and cool (21, 85, 109, 123).
(2) Irrigation
In the cultivar 'Grand Alexander', a direct corre-
lation was found between the frequency of irrigation and the
incidence of brown core (98).
(3) Fertilization
Heavy nitrogen fertilization and high manuring are
reported to increase the susceptibility of the fruits to
brown core (21, 37, 85, 105, 109). Nitrogen exerted some
effect other than merely influencing the growth of the fruit,
since there was no correlation found between cell size and
incidence of brown core (21, 57).
(4) Limb and fruit shading
Smock (106) found that limb shading consistently
increased susceptibility to brown core. However, he found
little to no effect due to fruit shading on the development

of the disorder. The susceptibility to brown core in this

case seems to be more related to the position of the fruit
in the tree, since Jackson (57) found more brown core in

fruit from "inside" than fruits from "outside" the tree.

b. Effects of Pre-storage Factors
(1) Fruit source
Brown core is known to vary from orchard to orchard
(h, 13, 28, 37, 63) and from area to area and from tree to
tree in the same orchard (37). While these differences are
recognized, no fully satisfactory explanations have been
advanced for them.
(2) Fruit maturity
Most of the researchers on fruit storage present
data showing that the more mature the fruits are at harvest
time, the less brown core they develop during storage (26,
37, 75, 81, 85, 105, 107, 109). However, some researchers
obtained opposite results in which the more immature fruits
were less susceptible (63, 126), while others had inconsis-
tent results (9: 13, 57). Fidler and North (28) stated that
the influence of maturity of the fruit at the beginning of
the storage period depends on the variety.
(3) Delayed storage
It has been suggested that a five day delay before
storage would greatly decrease the amount of brown core (90).
For the 'Starking Delicious' cultivar a #8 hr. period was
also reported to reduce the incidence significantly (75).
Other studies have shown that delayed storage did reduce the

intensity of the problem but that this finding had little

practical application (79. 106, 109). A delay in storage of
4 to 6 days at 380C has also been reported to eliminate the
problem in 'Spartan' and 'Golden Delicious' apples (87).

(A) Applied chemicals

Phorone (2,6 — dimethyl - 2,5 - heptadien - h - one)
markedly reduced brown core when fruits were exposed to
vapors (0.25 to l g. per 25 fruit) during storage. The
action of phorone in reducing brown core was found to be
reasonably specific since a large number of compounds with
a similar structure to phorone were found to have no effect
(95). ‘

GA3 had little to no effect in controlling the dis—
order (95) when applied as a post-harvest dip or injection
into the fruit.

B - 995 (N-dimethylaminosuccinamic acid) when sprayed
once during the growing season (2,500 ppm B-995) markedly
increased brown core in stored fruits (96).

DPA (diphenylamine) applied as a pre-storage dip
(1,000 ppm) also reduced the incidence of brown core (71),

but no explanations were advanced.

c . Effects of Fruit Factors
(1) Fruit size
Larger fruits have long been recognized as more sus-
ceptible to brown core (A). Light crops, which produce big-
ger fruits, or fruits from trees heavily fertilized with
nitrogen, tend to develop more brown core during storage

(4, 37, 109). However, Wilkinson (123) stated that the

available evidence suggests that fruit size, as such, is not
a factor influencing brown core.
(2) Fruit color

Fisher and Porritt (37) have noted that poorly
colored fruits were more likely to have brown core at the
end of storage. Smith (105) observed that fruits with the
highest percentage of green and lower percentage of yellow
as ground color developed more brown core during storage.
It seems that poor color is an indication of more immature
fruits and therefore more susceptible to develop the problem.

(3) Seeds

The effect of seed number in the fruit on brown
core was first noticed by workers in Canada (81). They
demonstrated that brown core is related to a particular phase
of physiological activity of seeds. Brown core did not
develop when the seeds were killed by controlled dosages of
irradiation. However, the same type of treatment failed to
prevent brown core in New Zealand apples (81). Came (11),
working with apples in France, suggested that the seeds do
not exert any effect on the development of brown core; on
the contrary, the disorder had a detrimental effect on the

seeds, since it rendered the embryos unable to germinate.

(1. Effects of Storage Factors
(1) Temperature
The majority of the reports concerning brown core
in apples, especially in 'McIntosh‘ fruits, implicate low

temperature of storage as being the main cause and several

investigators suggest that brown core is essentially a low
temperature disorder (4, 37, 81, 85, 106, 107, 109). How-
ever, inconsistent or even contradictory results can be found
in the literature concerning the role of temperature in
causing brown core. Meheurick and Porritt (75) found little
or no effect due to temperature on brown core and Fidler and
North (28) stated that this injury had no correlation with
temperature of storage between 0°C (32°F) and 3°C (37.4°F)
and also that the cultivars 'Fameuse' and 'Baldwin' had less
brown core at 0°C than at 2°C (35.60F) to 4°C (39.2°F).
(2) Humidity

In an experiment with fruits subjected to different
degrees of water loss, Wilkinson (124) observed that the
percentage of brown core was greater in apples which had
lost the most weight, and so it was suggested that permea-
bility of the fruit skin to gaseous exchange decreases rapidly
when evaporation takes place and that the greater effect of
early water loss may be due to a physical effect resulting
in restricted ventilation of the intercellular air spaces
(123). However, Scott and Wills (94) noted that there was
less brown core when apples were stored in the presence of
compounds which absorbed water and furthermore, while the
loss of water reduced brown core, the addition of water to
the fruit increased the incidence of the disorder.

Forsyth and Lightfoot (40) also noted that high humidity

would increase considerably the amount of brown core in

stored 'McIntosh' fruits.

9

(3) Carbon dioxide and oxygen levels

Fidler and North (28) are among those who strongly
support the view of brown core as being caused by 002. In
fact, they presented evidence from storage trials over a
period of ten years in which the incidence of brown core was
always more severe in the presence of CO2 than in its absence,
although the incidence varied greatly from year to year and
from one orchard to another. This same view is expressed in
a number of other reports (26, 27, 75, 94, 125). However, it
was recognized by Fidler and North (28) that brown core is a
senescence problem since it only occurs toward the end of
the storage period.

Some evidence presents brown core as being a conse-
quence of an association of low temperature and high C02
atmospheres (over 3%). especially in the presence of high
02 levels (67, 106, 112).

Eaves 33 a1. (19) found a high susceptibility to
brown core in fruits from trees with low nitrogen, as deter-
mined by leaf analysis, stored in presence of CO2 at all 02
concentrations above 2.5%. In contrast, the disorder appeared
to be reduced by C02 in the high nitrogen fruit.

The benefits of CA storage on controlling this dis-
order have been widely recognized (81, 85, 106-109, 117) and
Pierson.§3 a1. (85) attribute to this fact the promotion of
CA as the principal method of storing this variety.

Controlled atmosphere storage of apples as it is

being investigated nowadays, namely low levels of O2 (2.5-3%)

IO

and almost no CO2 in the storage environment (49), would seem
to have a protective effect in relation to the incidence of
brown core, since evidence suggests that apples will develop
less injury at low 02 levels (26-28, 125). However, low 02
by itself was not sufficient in preventing the appearance of
the problem (86). In addition, some inconsistent results
were found in which brown core was aggravated, but not nec-
essarily caused, by storage in controlled atmospheres (13,29).
(4) Length of the storage period

The fact that apples show the injury only after a
certain period of storage (generally 3 months) led some
researchers to state that brown core is a senescence dis-
order (26, 28, 125). Three interesting hypotheses were
developed to try to explain this phenomenon (28). The first
accounts for an increase in susceptibility to the injury as
fruit ages: the second relates the disorder to the duration
of the exposure to the adverse conditions causing it; and
the third suggests that the internal concentration of CO2
increases as the apples age.

There is no available evidence on the question of
changing susceptibility. However, Fidler and North (28)
stated that the rate of production of CO2 by 'Cox's Orange
Pippin' apples, stored at 3°C and 5% C02 z 16% 02, rises
slowly after about six weeks in storage and that the same
happens in C02 - free atmospheres, but‘uuarise is progressive-
ly delayed as 02 concentration is reduced. Therefore, if we

assume no change in permeability of the fruit, the internal

11

C02 concentration must increase during storage. If CO2 is,
in fact, the adverse factor in causing brown core then this
evidence fits perfectly the second and third hypotheses ad-
vanced by Fidler and North (28). Furthermore, there is some
published evidence (Kidd and West, 1939 cited in 28) that the
permeability of the fruit to C02 decreases after the climac—
teric. If this is so, it will contribute to the rise in the
internal C02 concentration.

(5) Ethylene levels

The effect of atmosphere of storage containing low
levels of ethylene on brown core was first noticed by For—
syth gt a1. (39). They found significantly less disorder
in 'McIntosh' fruits after storage for 189 days at 3.300 in
chambers containing KMnOu. Forsyth and Lightfoot (40) con-
firmed this observation with 'McIntosh' apples.

More recently Scott and Wills (94) found no corre-
lation between brown core and the ethylene level of the
storage environment in an experiment in which apples were
stored in plastic bags in the presence of KMnOu at 10°C. It
is interesting to notice that this temperature is considered

to be safe with respect to brown core incidence (107, 109).

e. Effects of Post-storage Factors
(1) Holding period at elevated temperatures after storage
Brown core has been noticed to increase in severity
and amount after the apples are removed from cold storage

and placed in warmer temperature (28, 37, 81, 85) and this

12

feature of the disorder seems to be very consistent since it
happened with fruits grown in the United States (85), in New
Zealand (81), in British cultivars (28) and also in Canada

(37).

2. Association of Brown Core to Other Disorders

The association of brown core with the disorder known as
stem-cavity browning has long been noticed, first by Ras-
mussen (90) in 1937 who described briefly a browning of the
skin of the stem cavity of 'McIntosh' apples and referred to
it as "stem—end breakdownfi and then by Smith (105) in 1942
who reported a similar condition as "stem-end rot". The
brief treatment by these authors caused these reports to go
largely unnoticed. However, McColloch (73) in 1966 finally
demonstrated the association of the two disorders and pro—
posed the possibility of them having a common cause.

Webster 23 al. (122) studied the stem-cavity browning
of 'McIntosh' apples as influenced by fruit size, position
in the blossom cluster and shape of the stem—cavity region.
They found apples from terminal clusters more susceptible to
the disorder than apples from lateral clusters. Larger
fruits also developed more stem—cavity browning as well as
fruits with shallow stem cavities.

Webster and Eaves (121) found that the incidence of
brown core and stem-cavity browning increased with increase
in fruit size. They confirmed their previous results, namely
stem-cavity browning being more severe in terminal apples than
in lateral apples and decreasing with increase in stem-cavity

depth.

(‘1‘

n)

13

Lougheed 23 a1. (70) recognized several features in
common for both disorders: the increase in incidence after
removal from storage; the highest incidence in immature
fruits; and considerable variation among years and locations.
However, they suggested that the physiological relationship
is not clearly defined and regarded the association of both

disorders as being a coincidence.

Low Temperature Breakdown

Low temperature breakdown (LTB) has been reviewed by
Faust gt a1. (21) and more recently by Wilkinson and Fidler
(125) in an excellent review.

It seems obvious for this particular disorder that the
cause is a low temperature of storage and the time of expo-
sure of the fruits to that low temperature (21, 26, 107,
125). However, a number of other factors militate to aggra-
vate the injury and will be mentioned here.

The fact that LTB varies greatly from year to year and
from location to location indicates that it can be influenced
by pre-harvest or orchard factors. Perring (84) observed
that LTB is less likely in apples with high levels of’K, P
and Mg than in apples with low levels of these elements.

An interesting feature of LTB is that the disorder
becomes worse if the apples are cooled when they are at a
critical physiological state, namely in the climacteric rise
(26) and this seems to be the origin of some contradictory
reports on the effect of delayed storage on LTB. Delayed

storage can either increase (63) or decrease (12) LTB,

14

depending on the physiological stage of the fruits when sub—
jected to the delaying treatment.

The composition of the storage atmosphere is known from
early days to have an effect on the extent and severity of
LTB (125). Accumulation of C02 to levels above 5% has been
reported to be detrimental to the quality and to increase
LTB (125). More recently, Knee and Bubb (63) found that
levels of 5% CO2 + 3% 02 instead of 8-10% 002 significantly
decreased the incidence of LTB in 'Bramley's Seedling' apples.

A decrease in susceptibility in LTB was found in Tas-
manian apples stored at low levels of 02, but this effect
was shown to be subject to seasonal variations in English
cultivars (125).

Humidity in the storage environment was also noted as
having an effect in LTB. High humidity is known to increase
its incidence while low humidity generally decreases the

incidence (125).

Alleviation of Brown Core andgLow Temperature Breakdown
Most storage trials are conducted at constant tempera-
tures, but it is possible to arrange an interruption of the
exposure to the low temperature by introducing one (interim)
or more (intermittent) periods at a higher temperature, or
to arrange a gradual reduction or a gradual increase of the
temperature during storage. The first two methods are per-
haps the less feasible in practice but are effective in
reducing the incidence of both low temperature breakdown and

brown core.

15

Smith (103) was probably the first to utilize interim
warming for storage of fruits. He found that low temperature
injury in plums was virtually eliminated by using an interim
warming treatment whereas storage at constant low tempera-
ture for an equivalent period of time resulted in extensive
injury. Subsequently a "dual temperature method" for storage
of plums was developed in which fruits were kept at 31°F for
5 days and then the temperature was raised to 45°F to 50°F
(101). The theoretical basis for this treatment derived
from the assumption that while capacity to ripen is impaired
sooner or later by low temperatures, the injury is not imme-
diate, or at least is not, in its earliest stages, irrever-
sible.

More recently, it was found by Smith (104) that an
interim warming on the 16th day at 65°F for 2 days resulted
in an extension of the period of storage in 'Victoria' plums,
regularly stored in 1% 02 and 34°F, by reducing the amount
of low temperature injury.

Following the early work with plums, the interim warm-
ing treatment was applied to peaches and nectarines (2) and
apples (32, 53, 80, 102, 103, 125).

Interim warming was proved to be an effective method of
controlling low temperature breakdown in apples (53, 102,
103, 125).

Intermittent warming treatment (in which the fruit
receives more than one warming period) has also been used in

controlling brown core in apples (67, 80, 81, 107).

l6

Padfield (80) reported a good control of brown core in
'Granny Smith' apples when fruits stored at 31°F were warmed
for three periods after 6, 9 and 13 weeks at 64-65°F for 2
days. Only one warming period (either at 6, 9 or 13 weeks)
was not useful in controlling the problem.

Landfald (67) also reported a reduction in brown core
when Scandinavian apples stored at 32°F were warmed for

periods of 5 days at 59°F.

Hypothesis Advanced to Explain Brown Core and Low Temperature
Breakdown

1. Brown Core

Despite the considerable amount of literature on brown
core, very few hypotheses have been advanced to explain the
appearance of the disorder. This is probably because there
is still a great deal of controversy concerning the cause of
this injury.

Basically, the disorder has been regarded as due to
senescence (Carne, 1958 cited in 28), as a form of low temp-
erature injury (81, 106, 107), or as a form of carbon dioxide
injury (27, 28). No hypotheses were presented for the first
two possible causes. However, Fidler and North (28) presented
their hypothesis on brown core as being caused by C02. The
evidence in the literature clearly supports this hypothesis
in view of the reports on brown core being aggravated by high
levels of C02, especially at high 02 levels. Most of the
time brown core was more severe in the presence of CO2 than

in COB-free atmospheres. Furthermore, it has been

17

demonstrated that the respiration rate of 'Cox's Orange
Pippin' apples at 3°C and 2% O2 is only approximately 60% of
the respiration rate of fruits kept at the same temperature
in air (28). Consequently, one would expect less CO2 accu-
mulation in apples preserved in low 02 concentrations.

Brown core was also tentatively linked with senescence
on the basis that apples will slowly increase their C02 pro-
duction throughout storage (28). Also, if permeability to
C02 decreases after the climacteric,2nssuggested by Kidd and
West, 1939 (cited in 28), one should expect an enhancement

of the internal C02 concentration as the fruits age.

2. Low Temperature Breakdown

Although it is generally accepted that LTB is caused by
an exposure of the fruits to a low temperature for a certain
period of time, there is not an agreement on how the low
temperature effect brings about the appearance of the dis-
order.

The first hypothesis was advanced by Plank in 1941
(cited in 103). He suggested that poisoning of the cells
could occur through the accumulation of a volatile product
of the metabolism. Several investigators have tried to prove
this hypothesis by linking LTB to the accumulation of several
volatile substances during the storage period. Wills 23 al.
(132) observed that increased rates of water loss, which
reduce susceptibility of 'Jonathan' apples to LTB, caused a
reduction in the level of acetic acid in the fruit. Increas-

ing the level of acid by the injection of acetic acid into

18

the fruit increased the incidence of the disorder. They
suggested that acetic acid could promote LTB and its removal
as acetate esters or free acid would reduce the disorder.
Wills and Scott (131) also found that apples injected
with acetate, mevalonate, malonate, formate, butyrate, ben—
zoate, caffeine, amytal, ATP and octanol showed an increase
in LTB after storage at -l°C. They suggested that isoprenoid
metabolism may be involved in the metabolic events leading
to LTB. It was also shown that fruits stored at -l°C re—
tained the greatest amount of acetic acid (129). At 0°C and
2.5°C the amount retained was also high, but at 5°C and 10°C
it was very low. The loss of butyl, isopentyl and hexyl
acetates was highest at 10°C and decreased markedly with
decreasing temperature. It was suggested that LTB does not
occur at higher temperatures because the increased loss of
acetate as esters results in a reduction of the amount of
acetic acid available to produce the disorder (129).
Acetaldehyde has also been shown to accumulate in fruits
subjected to low temperatures, however, Smagula and Bramlage
(100), in a recent review on the involvement of acetaldehyde
accumulation on physiological disorders of fruits, concluded,
based on a whole body of evidence, that its accumulation is
a result, rather than cause, of tissue disorganization.
Accumulation of non-volatile substances has also been
shown to occur in fruits showing LTB. Fidler and North
(33-35) have shown an accumulation of sorbitol in fruits
injured by low temperature but a causal connection was not

established.

l9

LTB also increased when geraniol and a number of inhibi-
tors of isoprenoid synthesis were injected into the fruit
after harvest (130).

Probably the most known hypothesis on the mechanism of
LTB is the one put forward by Hulme gt gt. (53). They found
a relationship between the accumulation of oxaloacetic acid
(0AA) in the tissue and subsequent development of LTB in
cold stored apples. A short interim warming treatment re-
duced both the accumulation of 0AA and intensity of LTB.

They suggested that LTB is caused by an interference in the
operation of the Krebs cycle in the tissue.

However, Fidler and North (31) have more recently demon-
strated that the disorder is not entirely dependent on 0AA
concentration since they found comparatively high levels of

0AA in fruits not showing LTB.

Organic Acid Metabolism

 

Organic acid metabolism, especially that of the inter-
mediates of the tricarboxylic acid (TCA) cycle, may be impor-
tant in physiological disorders such as brown core and low
temperature breakdown. The metabolism of organic acids in
fruits was reviewed by Ulrich (116) and in the apple fruit
specifically by Hulme and Rhodes (49).

Organic acids are widespread throughout higher plants.
Buch (7) listed 79 different nonvolatile, non nitrogen-con-
taining, carboxylic acids present in higher plants from
which 18 were present in the apple fruit, either in the pulp

or in the peel.

20

The organic acid composition of apple fruits varies in
both amount and type of acids according to variety and loca—
tion (60).

At early stages of the fruit development, quinic acid
accounts for more than 50% of the total acid content of the
cortical tissue (45). As the fruit matures the concentra-
tion and amount of this acid decreases rapidly (45), and it
was suggested that it may be oxidized to citric and malonic
acid (47). Malic acid reaches a peak 50-60 days after petal
fall and begins to decline (54), and at maturity it predomi-
nates, being reported to vary from 80% (66) to 95% (49).
Citric acid remains at a low and steady concentration through-
out fruit development (54).

Krotkov gt gt. (66) reported that while malic acid
decreased after harvest, "other organic acids" tended to
increase. And the same observation was made by Robertson
(93) who noted that "unknown organic acids" increased rapidly
compared to malic and citric acid through the climacteric
rise.

The metabolism of organic acids has been studied in
apples subjected to low temperature and CA storage because
of the physiological problems that may arise from such
preservation methods.

Kollas (64) found a much greater total acid concentra-
tion in 'McIntosh' fruits from CA (3% 02 + 5% CO2 at 38°F)
storage as compared to regular cold storage at 32°F after

6 months of storage. However, three acid peak fractions

21

were higher in fruits from air storage. We can interpret
these results in basically three ways: a) by supposing a
difference in the rate of depletion of the acids; b) by
supposing a greater production of the acids in CA storage;
or c) a combination of both.

Malic acid loss has been demonstrated to be independent
of oxygen since it was the same in air and in nitrogen over
a period of 80 days at 12°C (24). There is also evidence
that 'McIntosh' apples in 5% CO2 produce malic acid at sig-
nificant rates by fixation of 002 (l, 77). Thus it was
suggested that the higher malic acid content in apples stored
in CA atmosphere may have resulted from CO2 fixation (64).
Fidler and North (30) advocate that CA storage reduces the
rate of loss of acid from the fruit.

Hulme and Wooltorton (55) observed that citric acid in
the apple pulp increased from about 6mg/100g fresh tissue to
almost 10mg/100g fresh tissue over a period of 100 days at
15°C and that citric from the peel remained the same at
about 1.8mg/100g fresh tissue throughout.

An accumulation in both CA and air storage of oxalo-
acetic acid (OAA), pyruvic and -<-oxoglutaric acids was also
observed (53)- An interim warming treatment after 5 weeks
of storage for 5 days at 15°C was sufficient to lower the
level of the acids to their original concentration which
then started to accumulate again toward the end of storage.

14 14

Conversion of succinic acid - G into fumaric acid - C

was slightly less in apples kept in high CO2 atmosphere than

22

those kept in air (77).

It is generally accepted that CA storage and/or low
temperature storage can cause an accumulation of some
organic acids in apples (43, 53, 77, 99) as well as in other
fruits (56, 69, 74, 99, 120, 127). The acids reported to
accumulate are 0AA (25, 53, 74, 133) and succinic acid (43,
46, 56, 65, 88, 120, 127). However, Wills and McGlasson
(128) were not able to find any accumulation of 0AA, o<-keto-
glutaric and pyruvic acids during storage of 'Jonathan'
apples at -1°C.

Generally, it is assumed that high levels of CO2 in CA
storage would interfere in the activity of succinic dehydro-
genase (41, 61) causing succinic acid to accumulate. High

levels of CO also interfered with the activity of succinic

2
oxidase (89) and succinic-cytochrome C reductase (6).

The activity of citrate synthase was also inhibited by
low temperature and there was an accumulation of O<-keto
acids in banana peel (76).

High levels of succinic acid were found in apples
exposed to unusually high CO2 concentrations (up to 20%),
but only when the fruits were kept at low temperatures
(37°F) since high CO2 at 50°F did not cause succinic acid to
accumulate (46). It was noticed that the accumulation of
succinic acid was accompanied by carbon dioxide injury and
it was proposed that succinic acid poisoning was the cause

for CO2 injury (46). However, intentional application of

succinate to the peel of apple fruits was not sufficient to

23

cause COZ-associated peel injury in 'Golden Delicious' apples
(68). Furthermore, it was recently found that in the presence

of high CO transfer of fruits from 0°C to 21°C inhibited

2.
further induction of C02 injury but did not inhibit the
accumulation of succinic acid (43). This suggests that CO2
injury may be a low temperature and CO2 interaction and rules
out succinic acid as a sole causal agent.

Apple fruits decarboxylate malic acid oxidatively
(malate effect) to pyruvate (14, 38, 59, 62, 91) and subse-
quently to acetaldehyde and alcohol (10, 78, 91).

Dilley (14) demonstrated malic enzyme activity in
'McIntosh' fruits in the oxidative decarboxylation of malate
and postulated that the malate effect is a reflection of
metabolic processes concerned with pyruvic acid utilization
and that is perhaps regulated by the availability of oxidized
pyridine nucleotide.

Since the demonstration of the operation of the classi-
cal Krebs cycle-cytochrome oxidase respiratory system in cut
tissue and mitochondria from apple fruits (44), a great num-
ber of reports dealing with the metabolism of TCA cycle
intermediates in apple tissue have been put forward (20, 48,
50-52. 58. 91. 97)-

Worthy of mention is the "in vivo" inhibitory action of
0AA to all TCA cycle oxidations found in mitochondria of
potato tubers and mung bean hypocotyls (115) and the action
of CO2 in several reactions of the cycle (118, 119). CO2 at

18% stimulated malate oxidation about 10%; suppressed

24

°<—ketoglutarate, citrate and NADH oxidations about 10% and
suppressed fumarate, pyruvate and succinate oxidations about
32% in 'Richared Delicious' apple mitochondria (97). This

experiment suggests that CO can have a strong controlling

2
effect on apple mitochondrial activity. The suppressed
oxidations of succinate and citrate could explain the reported
accumulations of these acids under high 002. The effect of
CO2 on mitochondria was clearly not due to an effect on a

single enzyme, such as succinic dehydrogenase. Many enzyme

and to different degrees.

systems must be sensitive to CO2

Behavior of Apple Fruits Under Low Oxygen and Anaerobic
Conditions

 

Apples will shift their respiration from aerobic to
anaerobic (alcoholic fermentation or zymasis) when subjected
to very low levels or complete absence of oxygen leading to
an accumulation of considerable amounts of ethyl alcohol and
smaller amounts of acetaldehyde (22).

The extinction point for anaerobic respiration in many
tissues is below 2% oxygen (88). Early in the storage season,
in 'Newton Wonder' and 'Bramley's Seedling' apples, the
extinction point lies between 1 and 3% oxygen, but it was
noticed that the extinction point shifts to higher concen-
trations of oxygen later in the storage season, and even-
tually in senescent apples alcohol may accumulate even at
100% oxygen (113).

Fidler (23) observed that the capacity for anaerobic

respiration of apples would change over the storage period

 

 

25

and would follow the same seasonal course as aerobic respira-
tion. McLean gt gt. (72) observed a similar seasonal varia-
tion in the capacity for anaerobic respiration, but they con-
cluded that the loss in the capacity for anaerobic respira—
tion was due to the successive cycles of anaerobiosis exper-
imentally imposed previously on the fruits.

The carbon dioxide output in apples kept in anaerobiosis
is lower than those kept in air (18, 24, 35). The ratio of
anaerobic to aerobic CO2 production was shown to be lower in
fruits from CA storage than that of fruits from air storage,
indicating a conserving effect of the modified atmosphere on
the mechanism of aerobic CO2 production (18). The ratio of
anaerobic to aerobic C02 production by 'McIntosh' fruits
increased during ripening at 20°C immediately following
harvest (18).

Fidler (24) observed that the presence of oxygen has a
conserving effect on the loss of carbohydrate (Pasteur effect)
and that the loss of carbon is greater in nitrogen than in
oxygen. He found that anaerobic respiration in apples is
identical, as far as the nature and quantity of the end
products are concerned, with the alcohol fermentation of
yeast. It was also observed that the amount of carbon
dioxide produced by apples in nitrogen is equivalent to that
which would be produced by oxidation of malic acid.

Loss of CO2 plus alcohol was equivalent to the loss of

carbohydrate plus acid (35).

 

 

26

The "1ocalization" theory of the Pasteur effect, which
assumes the localization of the glycolytic enzymes and sub—
strates in certain points of the cell, was demonstrated in
apples (5), but Barker and Khan (5) were not able to consis-
tently demonstrate the "activation/inactivation" theory of
the Pasteur effect, i.e., that hexokinase and phosphofructo-
kinase are activated in anoxia and inactivated in air.

Hulme (45) showed that oxaloacetic, pyruvic and °<Z-keto—
glutaric acids would decrease from their initial levels to
very low levels when apple fruits were kept at 15°C under
nitrogen atmosphere during five days with, presumably, an
accumulation in their precursors since, on readmission of
air, there was a rapid and temporary burst in their rate of
production.

The effects of periods of anaerobiosis on the storage
of apples can be detrimental in other respects, apart from
the accumulation of alcohol. Anaerobiosis can increase the
incidence of brown core and breakdown (36). Apple scald was
also found to be greatly induced by deliberate anaerobiosis

(17. 72)-

MATERIALS AND METHODS

Fruit Material

Fruits of the 'McIntosh' cultivar (Mgtgg domestica cv.
McIntosh) used in the first experiment were obtained from
four different locations comprising four different lots.
Lots 1 and 2 were obtained from two different orchards at
the Graham Experiment Station in Grand Rapids, lot 3 from a
commercial orchard at Laingsburg and lot 4 from another com-
mercial orcahrd at Leslie, Michigan. Fruits from all lots
were pre—climacteric having less than 0.1 ppm internal
ethylene. For the second experiment only fruits from lot 1
were used.

The fruits were harvested and transported to the Horti-
culture Research Center on the same day where they were ran-
domized and placed in refrigerated storage (36°F) while

waiting for the other fruit lots. Harvest dates were as

follows:
Lot 1 - September 21, 1978
Lot 2 - September 26, 1978
Lot 3 - September 29, 1978
Lot 4 - September 29, 1978

For the third experiment, the 'Empire' cultivar (a
cross between 'McIntosh' and 'Red Delicious'), harvested on

October 3, 1979 from Graham Station and kept in hypobaric

27

 

 

28

storage at 40 mmHg and 31.5°F for 34 days, was used.

Treatments

 

1. First Experiment

The first experiment was carried out in a commercial CA
(Controlled Atmosphere) storage facility. The one chosen
was Bull's Storage located at Casnovia. The cold storage
control treatments for this experiment were kept in refrig-
erated storage rooms at the Horticulture Research Center,
Michigan State University (MSU) at 32°F and 36°F in air.
The temperatures and controlled atmospheres available at

Bull's Storage were 32°F and 36°F with 3% 02 + 3% 00 The

2.
fruits were transferred to the definitive storage temperatures
and atmospheres on October 18 and the treatments were as
follows:
(1) 32°F, 3% 02 + 3% CO2 continuously
(2) 32°F, 3% 02 + 3% CO2 w/interruption at 3 months
for 2 days at 68°F
(3) 32°F, 3% 02 + 3% CO2 w/interruption at 3 months
for 14 days at 32°F
(4) 36°F. 3% 02 + 3% CO2 continuously
(5) 36°F, 3% 02 + 3% CO2 w/interruption at 3 months
for 2 days at 68°F
(6) 36°F, 3% 02 + 002 w/interruption at 3 months
for 14 days at 36°F
(7) 32°F in air continuously
(8) 32°F in air w/interruption at 3 months for

2 days at 68°F

 

 

 

29

(9) 36°F in air continuously
(10) 36°F in air w/interruption at 3 months for

2 days at 68°F

One box containing 150 fruits, approximately, was used
for each of the four lots comprising a total of approximately
600 fruits per treatment.

Fruits from the interim warming treatments were removed
from storage after 3 months and placed at room temperature
(68°F) for 2 days and then returned to their original storage
conditions. Some of the treatments were only interrupted at
3 months with respect to the storage atmosphere but remained
at the same storage temperature for 14 days and then were
returned to the original atmospheres. The CA control treat—
ments were kept continuously at 3% 02 + 3% CO2 at 32°F and
36°F.

Fruits of all treatments remained in storage for 7

months.

2. Second Experiment

For the second experiment only fruits from the lot 1
were used. A box containing approximately 150 fruits was
used per treatment and all the fruits were stored at the
Horticulture Research Center at MSU.

For the CA treatments, gas tight aluminum drums with a
volumetric capacity of approximately 1.7m3 were used and the
desired atmospheres were achieved by flushing initially or

upon atmosphere re-establishment with nitrogen until the

30

desired gas levels were reached. The oxygen and carbon
dioxide levels were monitored daily with an Orsat gas analy-
zer. Fresh air was admitted whenever the oxygen dropped
below the desired levels. Atmospheres containing 1.5 and

3% I 0.2% of 0 were used in the CA treatments. The carbon

2
dioxide levels were kept below 1% with the aid of dry lime
placed in the interior of the CA chambers. The chambers were
kept at 32°F and 36°F.

The hypobaric storage treatments were maintained in a
cylindrical tank with a volumetric capacity of approximately
1.16m3 placed inside of a storage room at 32°F and hence were
"jacket" cooled (Fig.1). The storage pressure (50 mmHg) was
achieved by withdrawing the air continuously with a Kinney
rotary oil seal vacuum pump located outside the storage room
and at the same time admitting ethylene-free air saturated
with water vapor through a humidifier maintained at the
reduced pressure to avoid dehydration of the stored fruits
(l5, 16). The coming air was regulated to the desired pres-
sure by a Fisher type 98 LD vacuum regulator and the flow of
the air through the system was throttled by an exhaust valve
leading to the vacuum pump. The humidifier was kept outside
the storage room and had a constant water level regulated by
a Sensall Model 501 ultrasonic liquid level switch which
activated a solenoid valve to admit filtered and de-ionized
water whenever necessary (Fig.1).

The fruits were placed under the treatment conditions

on September 25. The interim warming treatments were made

31

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by interrupting storage after 3 months and transferring the
fruits to air for 2 days at 68°F and then returning the
fruits to their original storage conditions. A period of
approximately 5 hours was necessary to warm the fruits to
68°F. Some of the treatments consisted of interrupting the
storage atmosphere but maintaining fruits at the same storage
temperature for 14 days after which they were returned to
their original storage conditions. There were CA, hypobaric
and common refrigeration control fruits which were kept at
32°F and 36°F continuously. The treatments were the following.

(1) 32°F, 3% 02 continously

(2) 32°F, 1.5% 02 continuously

(3) 36°F, 1.5% 02 continuously

(4) 36°F, 3% O2 continuously

(5) 32°F, 3% O2 w/interruption at 3 months for
2 days at 68°F

(6) 32°F, 1.5% 02 w/interruption at 3 months for
2 days at 68°F

(7) 36°F, 1.5% 02 w/interruption at 3 months for
2 days at 68°F

(8) 36°F, 3% O2 w/interruption at 3 months for
2 days at 68°F

(9) 32°F, 3% O2 w/interruption at 3 months for
14 days at 32°F

(10) 32°F, 1.5% 02 w/interruption at 3 months for
14 days at 32°F

(11) 36°F, 1.5% 02 w/interruption at 3 months for
14 days at 36°F

(12) 36°F, 3% O2 w/interruption at 3 months for
14 days at 36°F

33

(13) 32°F, 50mmHg continuously

(14) 32°F, 50 mmHg w/interruption at 3 months for
2 days at 68°F

(15) 32°F, 50 mmHg w/interruption at 3 months for
14 days at 32°F

(16) 32°F in air continuously

(17) 32°F in air w/interruption at 3 months for
2 days at 68°F

(18) 36°F in air continuously

(19) 36°F in air w/interruption at 3 months for
2 days at 68°F

All the treatments remained in storage for 7 months. In
the hypobaric chamber, a malfunction of the humidifier caused
the fruits to remain submerged in water for a few days prior
to the end of the experiment.

3. Third Experiment

Thirty fruits of the 'Empire' variety ranging from 111
to 188 grams were divided into two lots of fifteen fruits
and each lot subjected to a different atmosphere treatment.
Each fruit was placed in individual respiration chambers with
a volume of 18 m1 and a flow rate in the range from 18 to
20 ml/min. The temperature of the room was kept at 20°C
throughout the experiment. One lot received ethylene-free
air and the other received pure nitrogen.

At given intervals (0, 8, 32 and 128 hours) samples were
drawn consisting of three fruits from each lot and subse-
quently analyzed for organic acids. Samples of the gas
atmosphere inside the respiration chambers were also taken

at periodic intervals for CO2 analysis.

34

Fruit Maturity at Harvest

The maturity of the fruit lots was determined following
harvest by measuring the internal concentration of ethylene
of 10 fruits from each lot. One ml gas samples were taken
from apples submerged in water and injected into a Varian
aerograph series 1700 gas chromatograph which used nitrogen
at 60°C as the carrier gas and was equipped with a 2mm x 1m

column of activated alumina and a flame ionization detector.

Fruit Firmnegg

 

Fruit firmness was determined with an Effe-gi fruit
tester equipped with a probe of 11.1mm (7/16 inch) diameter.
Determinations were made at the beginning of the storage,
after 3 months of storage, at the end of the interruption
periods, at the end of the storage period, and after 7 days
at 20°C following storage. At the beginning of the storage
period, 50 fruits per treatment were used and two measure—
ments were made on opposite sides of the fruits. After 3
months of storage and the end of the interruption periods,
10 fruits per treatment were used and 4 measurements were
made per fruit. At the end of the storage period and after
7 days following storage, 20 fruits per treatment were used
and 2 measurements were made on opposite sides of the fruits.
In the case of 2 measurements per fruit, one of the measure-

ments was taken in the blushed side of the fruit.

Internal and External Disorders
Fruits were examined for disorders after 3 months of

storage and after the interruption periods using 10 fruits

35

per treatment and at the end of the storage period and after
7 days following removal from storage using 30 to 90 fruits
per treatment. A cut was made longitudinally in each fruit
and both surfaces were visually examined. Fruits were then
classified into different categories.
1. Brown Core
Brown core was characterized by a browning of the corti-
cal parenchyma in the intervascular region. This is gener-
ally a very characteristic arrow-shaped discoloration in the
core region. The browning sometimes extended beyond the core
region. Four arbitrary categories were chosen as follows:
None - Fruits completely sound.

Slight

Brown core hardly discernible. Only

identified by a trained person.

Medium - Brown core in a level as to be easily
identified by a non-trained person,
but not in a level as to render the
fruit unmarketable.

Severe - Brown core in a level as to render the

fruit unmarketable.
2. Low Temperature Breakdown
Low temperature breakdown was characterized by an evenly
distributed discoloration of the whole cut surface of the
fruit. Sometimes a clear halo of unaffected tissue was
noticed beneath the skin. The same four categories as above

were used to classify this disorder.

36

3. Senescent Breakdown

This disorder, also known as internal breakdown, was
characterized by the softening and crumbling of the flesh
and by the eventual browning of the affected area. The dis-
order was classified in four categories as described above.
4. Scald

Superficial scald was identified by the characteristic
browning of the fruit peel which became worse following
removal from storage. In all cases, only the peel of the
fruits was affected, the underlying tissues remaining sound.
The fruits were classified according to the degree of inci-

dence of the disorder using the same four categories as above.

Titratable Acidity

Titratable acidity was determined prior to storage,
after 3 months of storage, after the storage interruption
periods and at the end of the storage period.

For the initial determinations (prior to storage) 12
fruits per lot were used and for the subsequent determina-
tions 5 fruits per treatment were used. Apples were peeled,
cored, and sliced and the pieces were randomized. Fifty
grams of tissue were weighed and placed in a Waring blendor
with 100ml of distilled water. The fruit tissue was blended
for one minute at full speed and filtered on a Buchner filter
flask using two layers of milk filter discs. An aliquot of
25ml was titrated against a standardized solution of 0.1N
NaOH. The titratable acidity was calculated as percentage

of malic acid.

37

Organic Acids

1. Organic Acids Extraction

The method used for organic acids extraction was especially
developed for the purpose of the present study and is herein
described.

A 50 gram sample of apple pulp tissue was blended with
100ml of boiling absolute ethanol in a Waring blendor for 5
minutes at full speed. The slurry was filtered through two
layers of milk filter and two layers of Whatman's filter
paper no. 2 in a Buchner filter flask to remove cell wall
material and the coagulated pectins. The ethanol was then
eliminated in a rotary evaporator (Rotavapor, Buchi) at 20°C
and the remaining aqueous extract was made basic to pH 8.5
with concentrated NHQOH and passed over a column of 1.5 x
30cm packed with 18ml of a precycled (12) strong anion
exchange resin (Amberlite IRA-400 grade l-X8) in the acetate
form to avoid degradation of the sugars (8).

The flow rate was maintained in the range from 0.5 to
lml/minute. The resin with the absorbed organic acids was
washed with 10 bed volumes of distilled water to remove all
the remaining sugars and then was eluted with 40ml of 1N HCl.
The eluate was then concentrated in a rotary evaporator at
35°C to an appropriate volume to be filtered and injected
in the chromatographic system. Using this method of extrac-
tion, recoveries very close to 100% were obtained when samples

were spiked with known amounts of standard acids.

38

Hydrochloric acid present in the eluate made the final
concentration process difficult, but addition of concentrated
NaOH was found to facilitate the process and therefore the
acids were analyzed in their salt form.

2. Organic Acids Analysis

For the analysis of the organic acids present in the
samples a modification of the method suggested by Waters
Associates, Inc. was used.

The High Performance Liquid Chromatograph (HPLC) system
consisted of a Waters Associates solvent delivery pump
(model 6000A), a Waters Associates Universal liquid chroma-
tograph injector (model U6K), a Waters Associates Differen-
tial Refractometer detector (model R401) and two/u BONDAPAK
C18 columns (Waters Associates, Milfbrd, Mass.).

The mobile phase was a buffer solution of 0.1M NHquPOu
with pH adjusted to 2.5 with H3P04 and the flow rate main—
tained at 1.4m1/min. with an operational pressure in the
pump of 3000psi. This procedure gave a better separation of
the acids, a better response in terms of peak height and was
faster than the procedure suggested by the manufacturer of
the equipment.

With this procedure, all of the organic acids of interest
present in the samples could be analyzed in 9 minutes.

The standards used were acids of high purity obtained
from Sigma Chemical Co. and a typical chromatogram of the

standards can be seen in Figure 2.

39

COLUMN two psondopok CIB
MOBILE PHASE 0.1M mango4 pH adjusted cozs w/H3P04
now RATE 1.4 ml min
PRESSURE 3000 psi

 

 

 

DETECTOR Refractometer E
.3

3

l

i

§ 3

l 3

l o
i .2
: §
.25 l 3
o 3 0
E I .3 2
3 l .. I
to- I .3 .8
fl ’ °

 

 

 

 

-.__ ___,J l U

9 3 7 6 5 4 3 2 ‘
TIME (min)

1
I
l
l
l
.1"- injection

Figure 2. Separation of the tricarboxylic acid cycle acids
by HPLC.

40

The peaks appearing in the chromatograms of the samples
were measured for retention time and compared with the reten-
tion time of the peaks from the chromatograms of the standards
and also samples were mixed with authentic organic acids to
additionally confirm the identity of the acids.

This system provided qualitative and quantitative analy-
sis for malic, citric, succinic and fumaric acid in the apple
extracts. Oxalacetic acid could not be quantified because
of the appearance in the chromatograms of the samples of an
unidentified peak which exceeded by far the normal concen-
tration of this acid in the fruit. There was an overlap of
the acetate peak (from the anion exchange column) with the
peak of'-<-Ketoglutaric acid and therefore it also could not

be quantified.

Carbon Dioxide Determinations

Gas samples were taken from the individual respiration
chambers, previously described, with a 2cc syringe through
septa located at the lid of the chambers and analyzed in a
gas chromatograph equipped with a thermal conductivity detec-
tor (Carle Instruments. Inc. GC 8700) using Helium as carrier
gas.
Statistical Analysis

Statistical analysis of data was by analysis of variance
procedure. Means comparison was by Duncan's multiple range
test.

To analyze the effect of the treatments on the physio-

logical disorders a procedure was developed as follows:

Ln

For the category "none" an arbitrary value of one was assigned,
for "slight" a value of three, for "medium" a value of 6 and
for "severe" a value of 10. The number of fruits going into
each category was multiplied by the category value and the
results were summed and divided by the total number of fruits
used for the observation. This procedure generated a single
value for each physiological disorder at each observation

period which was then used for statistical analysis.

RESULTS

First Experiment

The first experiment tested the incidence of physiologi-
cal disorders under commercial CA storage conditions and in—
cluded fruits from 4 orchards.

The results for firmness and titratable acidity expressed
as percentage of malic acid of the first experiment can be
observed in Table 1.

After 7 months of storage, firmness was retained better
in CA storage even when a temperature of 36°F was employed
as compared to air storage. Interruption treatments didn't
significantly affect firmness and the air stored fruits which
received a 2 days at 68°F interruption had, in fact, a higher
firmness after 7 months of storage, but this effect was not
noticed in CA stored fruits. After the holding period, the
same trend was observed; namely, CA stored fruits were firmer
than air fruits. No statistical significance was found for
temperature and interruption effects or for the temperature/
interruption interaction.

The total acidity content of CA stored fruits was sig—
nificantly higher after 7 months of storage than that of air
stored fruits. The acidity of the fruits from the storage

interruption treatments was generally higher than the acidity

42

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on» new ommnopm mo mapsoe m nmpmm moms who; mesoEPMonp :owpmsnpopsfl owmnopm HH< 3

 

43

 

 

 

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o ssa.o so m.m oo m.a sansoseapsoo use town
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s ssm.o on :.m one H.0H Amomo as name NV <0 soon
a mmm.o no e.a s m.oa sansoseapsoo so moon
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44

of the fruits from continuous storage and this effect was
found significant at the 2.5% level for the air stored fruits
but not for the CA stored fruits.

The incidence of brown core was higher in air stored
fruits than in CA stored fruits after 7 months of storage
(Table 2). Brown core was slightly higher in both CA and
air stored fruits at 32°F than at 36°F. No statistical sig-
nificance was found for this effect in CA stored fruits, but
in air stored fruits a significance at the 2.5% level was
observed for the temperature effect. Interruption of the
storage conditions had no effect on brown core development.
Significant differences (at 0.1% level), however, were
observed in the amount of brown core among the different lots
of fruits after 7 months of storage. After holding the fruit
in air at 20°C, brown core generally increased in all treat—
ments, and the same trend was observed with air storage
having more incidence of brown core than CA stored fruits and
the disorder being worse at 32°F than at 36°F. Interruption
of the storage environment had no effect on the final amount
of the disorder. Significant differences were also found in
the amount of the disorder among the different lots of fruits
(at 0.1% level).

The effect of the different treatments on the amount of
superficial scald was not found to be significant after 7
months of storage. For CA stored fruits, interruption of
the storage environment tended to increase scald (significant

at 1% level). This effect was not observed in air stored

45

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o om.a oo.H e 36.: .n.esm.m o mm.a o oe.a Amowo on name NV so moan
on am.a oo.H eoo ms.s .o.eoo.m o mm.a No me.H sansossapsoo sec sown
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peoanMHo noes: m:p£oe u now oonopm mmammw .nmochos. so smnmunomao Hwowwoaofimhnm .N manna

46

fruits which showed lower amounts of scald when previously
subjected to the storage interruption treatment. Different
sources of fruits had significantly different levels of scald
(at 0.1% level).

During the 7 day post-storage holding period scald
increased in all treatments. No marked benefit was evident
from the storage interruption treatments on the final amount
of scald. In fact, CA stored fruits which were interrupted
showed increased scald on holding after storage. Air stored,
which previously showed less scald when interrupted, showed
more scald after the holding period. Air stored fruits had
a significantly higher level of scald when kept at 32°F (at
5% level), but this effect was not observed in CA stored
fruits. The final amount of scald showed to be significantly
different (at 0.1% level) among the different lots of fruit.

Senescent breakdown was absent in all the treatments
following 7 months of storage. But after the holding period,
fruits stored at 32°F had more breakdown than those kept at
36°F (significant at 5% level) in both CA and air stored
fruits. Senescent breakdown was slightly more severe in
fruits kept in air at 32°F. No clear-cut benefit was observed
due to the storage interruption treatments and breakdown was
significantly different among the different fruit lots (5%

level) in CA stored fruits.

Second Experiment
The second experiment was conducted in order to gain

additional information on the factors involved in the

47

physiological disorders, especially brown core and low tem-
perature breakdown using the more precise and reliable
research facilities available at the Horticulture Research
Center at MSU. Hypobaric environment, apart from its excel-
lent prospects as a storage method, is a very good research
tool whenever one wants to study the involvement of ethylene
or other volatiles in a given physiological process, and
therefore, was included in the experiment.

Due to physical constraints we were unable to replicate
each treatment, which reduces considerably the scope of infer—
ence of the experiment, and this should be considered in
interpreting the results. Statistical analysis was conducted
by analysis of variance for the first 12 treatments (CA
treatments) which are arranged in a factorial experiment and
the significance was tested by the F test. The main effects
analyzed were temperature, 02 concentration and interruption
treatments. The air storage treatments were analyzed sepa—
rately but, perhaps due to the lack of replications and to
the small number of treatments, no significant differences
were found.

The fruits used in this experiment were in the pre-
climacteric stage at the beginning since their internal ethy-
lene concentration averaged 0.1ppm (average of ten fruits).

The results of firmness and titratable acidity expressed
in percentage of malic acid are shown in Table 3. After 3
months of storage firmness was higher in the fruits stored
at 32°F and 1.5% 02 (statistically significant at 0.1% level).

The interaction between temperature and oxygen level was also

48

 

 

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49

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meson oaaoehww
seaeaoe marmoseeas

sanoaqooosEhaa

A.o.eeoov m oases

50

significant at the 0.1% level suggesting a possible inter-
dependence of both factors up to that time in storage.

After 7 months of storage, oxygen was more efficient
than temperature in maintaining the firmness, since fruits
kept at 1.5% 02 had higher firmness values (significant at
2.5% level) regardless of the storage temperature. However,
the temperature effect was also significant at the 5% level.
The interruption treatments didn't affect the firmness enough
to be significant. After the additional holding period of
7 days at 68°F, however, only the fruits from the treatments
which had 1.5% 02 showed high firmness values (significant
at 5% level) and no effect due to temperature was observed.

No significant differences in acidity were detected
after 7 months of storage and also after the additional hold-
ing period of 7 days at 68°F. This was probably because of
the limited number of observations taken with the experimen—
tal set-up used.

Firmness 0f the hypobarically stored apples was consis-
tently high up to 7 months in storage as compared to firmness
of CA stored and air stored fruits. However, near the end of
the storage period a failure of the humidifier caused the
hypobaric chamber to be filled with water and this unexpected
problem, though promptly corrected, resulted in a very unfa-
vorable environment for the fruits for a short period of time,
and this is probably the origin of the lower levels of firm-
ness and acidity of hypobaric fruits, as compared to some of
the CA storage treatments. Fruits kept at 32°F and 1.5% 02

had the highest firmness throughout the experiment.

51

The results for physiological disorders can be seen in
Table 4.

No significant differences were found in brown core at
the end of the storage period for CA stored fruits. Brown
core was absent in hypobaric storage and slightly higher in
air stored fruits after 7 months of storage. However, after
the holding period at a high temperature, in which brown core
is enhanced, brown core was found worse in air stored fruits
and significantly higher in 3% 02 fruits as compared to 1.5%
02 fruits (significant at 2.5% level). Interruption of the
atmosphere in low 02 treatments yielded no clear-cut benefit.
Interruption for 14 days at low temperature had the least
brown core in low 02 treatments after the holding period.
Fruits from continuous hypobaric storage treatment had more
brown core than those fruits interrupted for 14 days at 32°F.
And interruption for 2 days at 68°F had the least brown core.
For the fruits kept in air the trend was similar in that
fruits that had been interrupted for 2 days at 68°F showed
less brown core after the holding period of 7 days at 68°F
than if held continuously in storage at 32°F.

Scald showed no significant difference among the treat-
ments for CA stored fruits after 7 months of storage. A
trend, however, was observed in which fruits kept at 3% 02
showed consistently more scald than those kept at 1.5% 02.

For hypobaric storage a browning of the peel resembling
scald was observed in the fruits after 7 months of storage

and was, therefore, rated as scald. This may be related to

52

 

 

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mass A mafia s mafia s mafia s

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.d mHnme

53

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A.o.eooov e oases

54

the flooding mishap near the end of the storage period. This
injury decreased after the holding period.

Air stored fruits showed a higher amount of scald as
compared to CA stored fruits after 7 months of storage.

After the holding period at room temperature, a greater
amount of scald was found in fruits kept at 3% 02 as compared
to 1.5% 02 (significant at 2.5% level) confirming the trend
observed after 7 months of storage. Scald appeared worse in
fruits kept at 36°F than at 32°F, however, this difference
was not found statistically significant.

Senescent breakdown was absent in low 0 storage fruits

2
after 7 months of storage, showing only after the holding
period at high temperature. At that point, fruits which were
kept at 36°F showed more senescent breakdown (significant at
5% level) as well as fruit from the 3% 02 treatments (signi-
ficant at 5% level). Interruption treatments showed no
effect on the level of senescent breakdown. In air stored
fruits the same temperature trend was observed, namely, fruits
stored at 36°F having more disorder than those kept at 32°F
after the holding period. Senescent breakdown was higher in
both observation periods in hypobaric storage and air storage
as compared to low oxygen storage.

No significant difference was found in low temperature
breakdown following 7 months of storage or after the holding
period in low oxygen or in air. However, fruits kept at 3%

02 showed a tendency to have more LTB after the holding

period.

55

Hypobaric storage, however, showed a greater amount of
LTB after the holding period when the fruits were kept con-
tinuously in storage and decreased when an interruption
treatment was used, with 2 days at 68°F being more efficient
in reducing the level of LTB than 14 days in air at low
temperature. However, as noted earlier, this damage may be
related to anoxia from a flooding mishap to the hypobaric

chamber.

Third Experiment

 

1. Organic Acids
Comparison of the results was done by the analysis of
variance and the differences evaluated by the F test.

No statistical difference was found in the amount of
organic acids between aerobic and anaerobic atmospheres when
the two sets of data were compared at the same observation
period throughout the experiment.

However, when two sets of data from the same treatment
(aerobic or anaerobic) were compared at different observa-
tion periods, some statistical significance was found indi-
cating changes in the amount of some acids during the exper-
imental period. The results for individual acids are des-
cribed below.

a. Malic Acid
The results from the High Performance Liquid Chromato-

graphy determinations of malic acid are shown in Figure 3.

56

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:popoo m mo ommuo>m so mpcomopmop psfion memo zoom .ooom pm monogamoepm
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57

Aerobic and anaerobic fruits behaved similarly in res-
pect to malic acid content during the experiment period and,
in fact, no statistical difference was found between the two
treatments.

Although the data suggests a decrease in the amount of
malic acid in both aerobic and anaerobic conditions over the
period of 128 hours, only anaerobic fruits were found signi-
ficantly different from the initial concentration at 1% level.
b. Citric Acid

The level of citric acid, in both aerobic and anaerobic
stored fruits, remained constant during the experiment period
(Figure 4). The data shows the anaerobic fruits with a
citric acid content consistently higher than aerobic fruits,
but no statistical differences were found between the two
treatments in any of the three observation periods and also
no statistical differences were found when the same treatment
was compared among the different observation periods, which
shows that the amount of the acid was not changed by any of
the treatments and that it remained constant throughout the
experiment period (128 hours).

c. Succinic Acid

The amount of succinic acid in aerobic and anaerobic
fruits showed a similar behavior (Figure 5). They initially
decreased slightly toward 8 hours and then increased toward
32 hours, aerobic fruits having a more pronounced increase
than anaerobic fruits. The level then remained constant

toward 128 hours and at the end of the experiment they had

58

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60

almost the same concentration.

In spite of the consistently higher amounts of succinic
acid under aerobic conditions, no difference was found
between the two treatments in any of the observation periods.
although the data suggests a more sharp increase in succinic
acid in aerobic fruits from 8 to 32 hours.

However, a significant difference was found at the 1%
level between the initial and the final concentrations under
aerobic conditions which shows an increase in the amount of
the acid over the period of 128 hours. A similar significance
was not found under anaerobic conditions. But if we compare
the concentration of succinic acid from 8 hours (minimal
inflection point of the curve) with the final concentration
(128 hours), then both the aerobic and anaerobic treatment
show a significant increase.

d. Fumaric Acid

The behavior of fumaric acid under aerobic and anaerobic
conditions was quite similar to the behavior of succinic acid
described above (Figure 6).

The inflection point of the two curves at 8 hours was
more pronounced than that of succinic acid and the level of
fumaric acid increased thereafter under both conditions
toward 128 hours. Again, the aerobic condition consistently
showed higher levels of the acid but no statistical differ-
ences were found in any of the observation points.

The increase in the amount of fumaric acid from 8 to

128 hours was more pronounced in the aerobic atmosphere than

61

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62

in the anaerobic atmosphere, but the increase was significant
in both cases.
e. Carbon Dioxide Production

Carbon dioxide production of 'Empire' fruits kept in air
or nitrogen at 20°C is shown in Figure 7. Each data point
represents the average of CO2 production of eight individual
fruits.

Data gathered eight hours after the fruits had reached
equilibrium in the respirometers shows a marked reduction in
CO2 evolution of the fruits kept in nitrogen as compared to
the CO2 evolution of the fruits kept in air. The anaerobic
CO2 output decreased even more toward 128 hours while aerobic
respiration increased until 56 hours and then held fairly
steady through 128 hours.

The ratio of anaerobic to aerobic CO2 evolution decreased
from 0.61 at 8 hours to 0.17 at 128 hours.

No visible symptoms of low 02 injury were observed in

the fruits kept under nitrogen up to 128 hours.

l5—

ml COZ/IrgJu'
r

 

63

 

 

/A\A_f Anglicbf #A
A

 

 

 

o
5 r—
o
I- N .
fl ; Anoeroluc
l- ‘C o
J l l l l l
O I 32 56 II III ‘II
HOURS
Figure 7. C0 evolution of 'Empire' apples kept in air or

nigrogen at 20°C. Each data point represents an
average of 8 determinations using individual fruits.

DISCUSSION

From a review of the existing literature, it becomes
clear that the task of interpreting the results of experi-
ments on physiological disorders of apples, such as the dis-
orders included here, is not an easy one. Smock (107) recog-
nized that there is considerable confusion in the literature
on the terminology of internal storage disorders of apples.
They presented as an example "internal breakdown", which is
a term widely used to cover all such problems. Many types
of flesh breakdown can only be described visually, and they
are therefore difficult to define and are sometimes given
different names in different parts of the world.

The terms "low temperature breakdown", "senescent break-
down", "brown core", and "C02 injury" are all used to des-
cribe brown or dead cortical tissue of pome fruits. The des-
criptive nature of these terms implies that the cause of the
disorder has been established, but this is usually not so.
Faust gt gt; (21) observed that it is not feasible to differ-
entiate consistently on symptoms alone and that critical
metabolic studies appear to be necessary for identification
of the cause of breakdown.

It is not surprising, therefore, that conflicting results

may very often be found since there is always the possibility

64

65

of different investigators using the same term to make refer-
ence to different problems having different physiological
origins.

First Experiment

The results of flesh firmness data obtained from CA
stored fruits after 7 months of storage show the remarkable
effect of low oxygen in the maintenance of the physical inte-
grity of the fruits up to that time. Firmness was higher in
CA fruits kept at 36°F than in air stored fruits at 32°F,
indicating their younger physiological stage at the end of
storage time. This effect was also noticed after the holding
period at 68°F. There are some evidences that fruits kept
under CA conditions have a younger physiological age at the
end of storage when compared with fruit of the same chrono-
logical age kept under air storage (18).

The total acidity content of CA stored fruits was sig-
nificantly higher than air stored fruits after 7 months of
storage. This behavior of CA stored fruits was also observed
by other authors (1, 31, 64) and it has been explained as due
to a greater production of organic acids (mainly malic) (1,
64), due to a slower depletion of the total acidity (31),
and due to a combination of both aspects (64). Although it
has been shown that 'McIntosh' fruits will fix CO2 into malic
acid at considerably high rates (1), there seems to be no
grounds to explain the noticed effect with this hypothesis
alone since it was observed in the second experiment (which

is going to be discussed next) that there was a higher total

66

acidity at the end of the storage when low levels of CO2
were used (below 1%) associated with low levels of oxygen
throughout the storage. The data gathered in this research
(both first and second experiment) indicates that CA storage
has an effect in retaining the total acidity or lowering the
depletion rate of the total acidity and, therefore, the
higher levels of acidity observed in CA stored fruits at the
end of the storage period may be a result of higher CO2
fixation into malic acid and lower consumption of malic acid
during the storage period.

Controlled atmosphere storage decreased the incidence
of brown core and this agrees with several other reports
(81, 85, 106-109, 117). The incidence of brown core was
significantly higher at the lower storage temperature in CA
stored fruits which adds more evidence for the hypothesis
that brown core is a low temperature-related disorder. It
has also been suggested that brown core is a form of carbon
dioxide injury induced after continuous exposure of the
fruit to high internal C02 concentrations and this worsens
after the climacteric rise when permeability to CO2 decreases
(28).

Since brown core incidence was significantly higher in
air stored than in CA stored fruits, it is tempting to rule
out 002 as the causal agent. Even though internal CO2 con-
centration was not evaluated, it is obvious that CA stored
fruits have higher internal CO2 concentrations throughout

storage than air stored fruits. However, it is also possible

67

that air stored fruits, because of their more advanced
physiological stage, would have gone through the climacteric
in storage much earlier than CA stored fruits and, conse-
quently, would have been exposed to critically higher inter—
nal C02 concentrations for a longer period of time than CA
stored fruits.

No significant effect on the amount of brown core
following storage was found due to the storage interruptions.
If we accept that the effect of interim warming in attenuat—
ing physiological disorders is due to the dissipation of some
accumulated metabolite(s) toxic to the tissue, then the
increase in the general metabolism brought about by both
storage interruptions was not sufficient to metabolize the
toxic material(s). Padfield (80), working with New Zealand
'Granny Smith' apples, was able to reduce brown core to a
low level after storage only when 3 interruptions at 64-65°F
for 2 days were used. Landfald (67) also had to apply
several warming periods of 15°C for 5 days to control the
disorder. Multiple interruptions of the storage period would
make this process infeasible on a practical scale. It would
have deleterious effects by aging the fruits causing soften-
ing, shortening of the storage life and senescence disorders.

The significant difference found in the amount of brown
core among the fruits from different orchards used in the
experiment suggests that the disorder probably has a pre-
storage origin. This fits the hypothesis of organic acids

accumulation since differences in the amount and type of

68

organic acids according to variety and location have been
reported in apples following harvest (60).

The level of superficial scald following 7 months of
storage was found to be independent of the storage tempera-
ture and atmosphere composition since no significant differ—
ence was found at that time. Scald increased during a warm
post-storage period and a greater incidence was found in air
stored fruits as compared to CA stored fruits. The higher
amount of scald in air stored fruits presents further evi-
dence of scald being an oxidative process (125). The storage
interruption treatments which were found to reduce the inci-
dence of scald (81), afforded no effect and, in fact, they
significantly increased scald in CA stored fruits after 7
months of storage and after the holding period. Fruits sub-
jected to the storage interruption treatments had a higher
level of exposure to oxygen than those kept continuously in
storage and consequently were more likely to exhibit higher
levels of scald after storage.

After the holding period, a significantly higher level
of scald was observed in air stored fruits kept at 32°F and
this agrees with previous reports that scald becomes worse
the lower the temperature (81, 125).

The fact that lots of fruits from different orchards
had significantly different levels of scald in both observa-
tion periods suggests that this disorder is an intrinsic
characteristic of the fruits and that it may also have its

origin in pre-harvest factors.

69

Wilkinson and Fidler (125) stated that senescent break-
down would develop further at high temperatures after the
fruit has been removed from storage. This characteristic
of senescent breakdown was also observed in this experiment
since senescent breakdown was absent after 7 months of
storage and developed further after a period of 7 days at
68°F. Any treatment that delays ripening would likely reduce
the amount of senescent breakdown (125). Therefore, it is
logical that CA storage would decrease the amount of senes-
cent breakdown and indeed there was less breakdown in CA
stored fruits as compared to air stored fruits. However,
senescent breakdown was significantly higher at the lower
temperature in both CA and air stored fruits. Normally, more
breakdown would be expected at the higher temperature (125),
since higher temperatures would tend to age the fruits more,
rendering them more susceptible to senescent breakdown.

The significantly different amounts of senescent break-
down among the different lots of fruits observed in CA
stored fruits indicate 'UMe different inherent ability of
the fruits to develop the disorder during storage and clearly

links it with pre-storage factors.

Second Experiment

After 3 months of storage, firmness was significantly
higher for fruits stored at 32°F and 1.5% 02, indicating the
younger physiological stage of those fruits. Flesh firmness
was reduced as the temperature and oxygen levels were

increased simultaneously.

70

After 7 months, however, oxygen level was observed to
be more efficient than temperature in maintaining firmness.
since fruits kept at 1.5% 02 had higher values regardless of
the storage temperature. After the holding period, only
oxygen level showed a significant effect in maintaining
firmness. A period of 7 days at 68°F was sufficient to erase
the temperature effect but not sufficient to erase the lower
oxygen level effect.

Fruits kept under low oxygen levels, either CA or hypo-
baric storage, had higher levels of total acidity after 7
months of storage and that confirms the results observed in
the first experiment. Fidler and North (31) stated that the
rate of loss of acid is reduced by reduction of the concen-
tration of oxygen, as long as the respiration remains aerobic,
and that it is also reduced by increasing the concentration
of carbon dioxide. The results observed confirm this hypo-
thesis.

After 7 months of storage, brown core was only absent
in hypobarically stored fruits. Fruits from low oxygen stor-
age had some brown core and air stored fruits had a slightly
higher incidence. At that period, 1.5% 02 level in CA was
f0und to have no significance to the amount of brown core,
and since the pressure used (50mmHg) in hypobaric storage is
equivalent to approximately 1.4% 02 relative to air at 1
atmosphere, we tend to attribute the absence of brown core
in hypobarically stored fruits to the low pressure in which

the fruits were stored. Ethylene (40) and CO2 (28) have

71

been implicated in the incidence of brown core and it is
possible that the observed effect was due to the removal of
both gases from inside the fruits in low pressure storage.

After the post-storage holding period, however, brown
core was worse in fruits kept under air storage and a com-
parison of the low oxygen storage treatments alone revealed
a significantly higher level of brown core at 3% 02 as com-
pared to 1.5% 02. This result clearly shows the involvement
of oxygen in the incidence of brown core.

No conclusive results were obtained for scald after 7
months of storage in the 10w oxygen treatments since no sig-
nificant difference was found at that period. Air stored
fruits showed higher amounts of scald as compared to low
oxygen treatments, confirming the results obtained in the
first experiment. In hypobaric storage, the browning of the
peel, resembling scald, was also considerably higher than the
scald present in the low 02 treatments; however, two evidences
suggest that the disorder observed in hypobaric storage was
not, in fact, scald. First, scald is known not to be a prob-
lem in hypobaric storage (15), and secondly, in the first
experiment, in all the treatments, and in the second experi-
ment, in most of the treatments, scald increased after the
holding period at the higher temperature and, in fact, this
is a known characteristic of the disorder (85). The peel
injury observed in hypobaric fruit decreased in all the three
treatments which would be a very unusual behavior of super-

ficial scald. The injury observed under low pressure may be

72

related to the inadvertent flooding that occurred near the
end of the storage period as a result of the humidifier
system failure.

After the holding period, scald increased and some air
storage treatments had almost all fruits affected by the
disorder. Fruits kept at 3% 02 had a significantly higher
scald level when compared to 1.5% 02. Those results confirm
the observations made in the first experiment and clearly
link scald incidence to oxygen level. This also adds evi-
dence to the hypothesis of scald being an oxidative process
(125)-

Senescent breakdown was absent after 7 months of storage
in fruits kept at low oxygen levels and this result is in
very good agreement with the younger physiological stage of
those fruits at that time. In hypobaric storage, however,
the observed levels of senescent breakdown are unusual and
again this may be due to the unexpected, unfavorable conditions
to which the fruits were subjected prior to termination of
the storage period. The higher level of senescent breakdown
in air stored fruits as compared to low oxygen stored fruits
confirms the observed results in the first experiment.

Fruits kept at 36°F in 3% 02 showed significantly more
breakdown after the holding period than those kept at 32°F.
This may be expected since at the higher temperature fruits
would be more advanced physiologically and consequently more
likely to be affected. Interruption treatments, with only

a few exceptions, increased the amount of senescent

73

breakdown and again, interruption treatments would tend to
age the fruits.

Air stored fruits showed considerably higher levels of
senescent breakdown in both observation periods as compared
to low oxygen treatments. Air storage allows fruits to ripen
more rapidly than in CA and hence would be least effective
in opposing the aging process.

No clear-cut results were obtained concerning the inci-
dence of low temperature breakdown (LTB) after 7 months of
storage and after the holding period for low oxygen and air
stored fruits. Fruits kept at 3% 02 tended to have more LTB
after the holding period. Interruption was without consis-
tent effect in low oxygen and air stored fruits because it
sometimes increased the problem in some instances while
decreasing it in others. In hypobaric storage after the
holding period, the interim warming did reduce the incidence
of LTB. And, it was more efficient in doing so than just
interrupting the storage atmosphere while fruits remained at
low temperature, indicating that a more drastic increase in
the metabolism is needed to metabolize the toxic substances

that may have accumulated.

Third Experiment
1. Organic Acids Extraction and Analysis

As observed by Palmer and List (83), the "official"
chemical methods for analysis of organic acids in foods are
simply too time-consuming fer most purposes and, in any case,

"official" methods are only available for a few acids (3).

74

A number of chromatographic methods have been developed
for determining organic acids in biological samples. The
ion exchange chromatography of organic acids from tobacco
plants using gradient elution as described by Palmer (82),
proved to be very useful when adapted with minor modifica—
tions to the analysis of organic acids of apples (55), and
this method was widely used till recently. However, in
those methods complete separation of all the acids of inter-
est is not achieved and quantification is achieved through
the laborious collection of numerous fractions and the sub-
sequent titrimetric analysis of each fraction with dilute
alkali to an indicator end point. More recently, several
methods using liquid chromatography (LC) or high performance
liquid chromatography (HPLC) for the determination of organic
acids have been developed (42, 83, 92, 111, 114). Those
methods proved to be very efficient and greatly decreased
the analysis time. However, when analysis is needed on a
large number of samples, even those methods can be time-
consuming, since the fastest method described in the litera-
ture takes approximately 25 minutes to separate all the acids
of interest investigated in the present work.

The separation of organic acids by the HPLC method
developed during this work proved to be quite fast (all the
TCA cycle acids of interest were separated in ten.minutes)
and extremely reproducible and was, therefore, a very useful
research tool in this experiment and it is likely that the

extraction method, developed in this work for organic acids

75

of apples, could be used for other biological material.

The purpose of this experiment was to observe the
behavior of some of the TCA cycle intermediates in apples
when subjected to anoxia since some information gathered so
far suggests that apples would accumulate some of those
intermediates under low oxygen conditions and at low temp-
eratures. Anaerobic conditions were employed at 20°C to
accelerate the process. It is recognized that anaerobiosis

at low temperatures may cause different effects.

2. Organic Acids
a. Malic Acid

Malic acid was depleted at the same rate in both condi—
tions, aerobic and anaerobic, at 20°C over a period of 128
hours. Fidler (24) observed that the presence or absence of
oxygen is without effect on the rate of loss of acid in
apple fruits. The results obtained here confirm the results
of Fidler (24) and show specifically that malic acid decreases
at the same rate in apple fruits under aerobic and anaerobic
conditions. More recently (31) it was observed that the
rate of loss of acid is reduced by reduction of concentra-
tion of oxygen, as long as the respiration remained aerobic.
b. Citric Acid

The level of citric acid remained constant over a period
of 128 hours at 20°C in both aerobic and anaerobic conditions.
Fruits under anaerobic conditions were consistently higher
in citric acid but the difference was not statistically sig-

nificant. The concentration of citric acid found and the

76

stability of the level agree with previous observations of
citric acid content in the pulp of apples kept at 15°C in
air (55), and additionally suggest that anaerobic conditions
do not change the normal behavior of this acid at least over
a period of 128 hours.
0. Succinic and Fumaric Acid

The behavior of both acids under aerobic and anaerobic
conditions was found to be very similar. Initially, both
acids decreased in air or nitrogen over the first 8 hours
and then increased toward 128 hours with the increase being
more pronounced from 8 to 32 hours. Levels of succinic and
fumaric acid in air were consistently higher than those
observed under anaerobic conditions, but the difference was
not significant. These results agree with previous observa—
tions by Handwerker (43) that oxygen concentrations between
3 and 21% did not affect the accumulation of succinic and
fumaric acid in apple fruits, and suggests that oxygen is
without effect in the accumulation of both acids during the
ripening process over a period of 128 hours at 20°C.
d. Carbon Dioxide Production

C02 evolution showed to be highly affected by anaerobio-
sis even after 8 hours of exposure of the fruits to the
nitrogen atmosphere, since a marked reduction in CO2 output

could be observed at that time. After 8 hours the CO evo-

2
lution by fruits in nitrogen continued to decrease gradually
and by 128 hours the ratio of anaerobic to aerobic CO2

evolution was 0.17.

77

The ratio of anaerobic C02 evolution also decreased in
'Sturmer Pippin' apples kept under the same conditions of
the present experiment over a period of 35 days (24) and a
ratio of 0.6 was observed by Dilley gt gtt (18) in 'McIntosh'
apples following harvest. The pattern of both aerobic and

anaerobic CO evolution observed in the present experiment

2
is similar to the one observed by Dilley gt gtt (18) for
fruits following CA storage.

The results gathered in this experiment concerning
organic acids suggest that 02 is without effect on the be-
havior of malic, citric, succinic and fumaric acid, and con-
sequently accumulation of succinic and fumaric acid would be
likely to occur regardless of 02 concentration. This con-
clusion is supported by the recent observation that oxygen
concentration between 3 and 21% did not affect the accumula-
tion of both acids in apples kept at 0°C (43).

However, the accumulation of succinic acid in CA storage
as to cause physiological disorders through the inhibition
of succinic dehydrogenase by high CO2 levels (41, 61, 77),
is by far greater than the one observed, since apples were
reported to accumulate as much as 21mg of succinic acid/100g
of fresh tissue (46).

It is more likely that apples would accumulate more
toxic organic acids. such as succinic (46), under high 002
concentration than under low 02 concentrations. However,
more metabolic studies are necessary to further clarify and

establish a cause and effect relationship between organic

78

acids accumulation and the incidence of physiological dis-

orders in stored apples.

SUMMARY

The effect of temperature, oxygen levels and storage
interruption on the incidence of physiological disorders and
on the acidity content of 'McIntosh' apples was investigated.

A study of the metabolism of organic acids and respira—
tion of 'Empire' apples in aerobic and anaerobic atmospheres
was undertaken and during that study a method of extraction
and analysis using High Performance Liquid Chromatography
was deve10ped for the quantification of the TCA cycle inter-
mediates.

The incidence of brown core was more pronounced at the

lower temperature and higher 0 concentrations and increased

2
after a holding period of 7 days at 68°F. A single storage
interruption was found not sufficient to attenuate the dis-
order.

Superficial scald was found to be somewhat influenced
by oxygen level, being worse in air and decreasing with de-
crease in oxygen concentration. It increased after the
holding period and storage interruptions tended to increase
the incidence of scald.

Senescent breakdown was increased by treatments that

increased the aging process of the fruit, such as high 02

concentration and higher temperatures. However, in one

79

80

experiment, low temperature of storage was found to increase
senescent breakdown.

Low temperature breakdown, which occurred in only one of
the experiments, was found not to be influenced by the fac-
tors investigated.

Oxygen was found to be without effect on the amount of
malic, citric, succinic and fumaric acid present during
ripening at 20°C. Malic acid decreased, citric acid remained
constant, and succinic and fumaric acid increased over a
period of 128 hours at 20°C in both aerobic and anaerobic
atmospheres.

Data are presented which show that the accumulation of
organic acids to toxic levels that takes place under CA
storage, and to which a role in physiological disorders has
been ascribed, may be due to the high CO2 levels present

under those conditions rather than 02 levels.

LI ST OF REFERENCES

9.

10.

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