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THE EFFECTS OF TEMPERATURE DIFFERENTIAL AND
SURFACTANT ON THE POSTHARVEST INFILTRATION

OF CALCIUM SOLUTION INTO JONATHAN APPLE FRUIT

By

Julian June-Ling Lee

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Horticulture

1979

ABSTRACT

THE EFFECT OF TEMPERATURE DIFFERENTIAL AND
SURFACTANT ON THE POSTHARVEST INFILTRATION
OF CALCIUM SOLUTION INTO JONATHAN APPLE FRUIT

33'

Julian June-Ling Lee

Deterioration of apple fruit quality during storage has been
shown to be related to low levels of calcium (Ca) in the fruit. Cur-
rent orchard and postharvest practices to increase fruit ca are not
completely satisfactory. The effects of temperature differentials
and surfactants on solution infiltration were examined to possibly
provide a direct and efficient means to enrich fruit with Ca.

Ca1cium.infiltration into mature 'Jonathan' apple fruit was
achieved by submersion of warm fruits in cold solutions of 2 and 42
Ca012. Temperature reduction of the submerged fruit decreased the
pressure of gases within the intercellular spaces (ICS). The cooling
solution was forced into the ICS of the cortex via open lenticels as a
result of the difference between the ambient pressure and the pressure
of the cooled internal gases. Solution infiltration was markedly en-
hanced by the addition of surfactant, L-77, in the cooling solution.

Since fruit Ca increase was proportional to the quantity of in-
filtrated CaCl2 solution as measured by fruit weight gain, the increase
in fruit Ca could be accurately estimated by the weight gain from a

known CaCl2 concn in the cooling solution. Increases as great as 16 mg

Julian June-Ling Lee

Ca/lOO g fresh weight were readily achieved, whereas increase of only
1 or 2 mg Ca/lOO g fresh weight have been reported for the convention-
al dip or drench methods.

The fruit Ca increase was increased by increasing CaCl2 concn in
the cooling solution, increasing initial fruit temperature, decreasing
cooling solution temperature, increasing submersion duration and de-
creasing surface tension of the cooling solution. Mbrphological char—
acteristics of the fruit, such as the quantity of open lenticels, are
proposed as additional factors affecting infiltration of the cooling
solution.

These results suggest that hydrocooling with a refrigerated CaCl2
solution would offer a practical means of Ca enrichment. An adequate
increase of fruit Ca to maximize the storage and market life of the

fruit could be achieved after harvest, but prior to fruit storage, in

this manner.

Dedicated to my parents, father-in-law and my dear wife.

Without any of them, this work would never have started.

11

ACKNOWLEDGEMENTS

The author wishes to express his sincere thanks and gratitude
to Dr. D. H. Dewey for his constant supervision, guidance, and un-
derstanding throughout these studies.

The author is also indebted to Drs. M. J. Bukovac, D. R. Dilley,
P. Markakis and L. F. Wolterink who provided valuable discussions,
reviewed the manuscript, and served as members of the guidance com-
mittee.

Grateful acknowledgement is also accorded to Dr. H. P. Rasmussen
for his valuable discussions and critical review of the manuscript,
and to Mr. V. E. Shull for his assistant in microphotography and the

operation of the electromicroprobe.

iii

Exp.
Exp.

Exp.
Exp.
Exp.
Exp.
Exp.
Exp.

Exp.

APPENDIX .

LIST OF FIGURES

INTRODUCTION .

EXPERIMENTS

I.
II.

III.
IV.
V.
VI.
VII.
VIII.

IX.

DISCUSSION . .

CONCLUSION . .

LIST OF TABLES .

LITERATURE CITED

TABLE OF CONTENTS

LIST OF APPENDICES O O O O O O O O O O O O O O O O O O O O O O O O

LITEMTURE REVIEW 0 I O O O O O O O O O O O O O O O O O O O O O O
OBJECTIVES OF mE STUDY 0 O O O I O O O O O O O O O O O O O O O 0

GENERAL MATERIALS AND METHODS . . . . . . . . . . . . . . . . . .

Fruit Weight Gain during Hydrocooling . . . . . .
The Distribution of Dye Solution in Apple Cortex
and the Anatomical Differnces between Open and
Closed Lenticels . . . . . . . . . . . . . . .
Dye as Tracer for CaCl in Apple fruit Tissue . .
The Effect of Surfactaats on Fruit Weight Gain .
The Effect of Submersion Duration and Initial
Fruit Temperature on Fruit Weight Gain . . . .
The Relationship between Fruit Weight Gain and
Ca Content in the Fruit . . . . . . . . . . .
Solution Infiltration into Apple Fruit Induced
by Partial Vacuum . . . . . . . . . . . . . .
The Effect of Fruit Temperature Reduction on
Solution Infiltration . . . . . . . . . . . .
The Relationship between Solution Infiltratio
and the Volume change of the Gas within the
Intercellular Spaces of 'Jonathan' Apple
Fruit . . . . . . . . . . . . . . . . . . . .

iv

Page

. l8
. 26
. 29

LIST OF TABLES

Table Page

1. Statistical evaluation of the effects of food dye (FD),
solution temperature (ST) and fruit temperature (FT) on
weight gain of 'Jonathan' apple fruit . . . . . . . . . . . . - 14

2. The effect of solution temperature (ST) and fruit tempera-
ture (FT) on mean weight gain of 'Jonathan' apple fruit

submerged for one hour . . . . . . . . . . . . . . . . . . . . l7

3. The mean X—ray intensity of Calcium and Chlorine in cortical

tissue of 'Jonathan' apple fruit hydrocooled in dye solu-

tion either with or without CaCl2 . . . . . . . . . . . . . . . 27
4. The effect of surfactants on mean weight gain of 21°C

'Jonathan' apple fruit during submersion in 4% CaCl2

solution at -1°C . . . . . . . . . . . . . . . . . . . . . . . 30

5. The mean weight gain of 'Jonathan' apple fruit under
simulated hydrocooling conditions (solution.temperature:
5 i 005°C, 270 CaC12 plus 0.1% 10-77) 0 o o o o o o o o o o o o o 32

LIST OF FIGURES

Figure
l. The surface of a 'Jonathan' apple fruit hydrocooled
in 4% CaCl2 solution tinted with blue food dye . . . . . .
2. Cross section of a 'Jonathan'apple fruit hydrocooled
in 4% CaCl2 solution tinted with blue food dye . . . . . .
3. Cross section of cortical tissue of 'Jonathan' apple
fruit hydrocooled in 4% CaCl2 solution tinted with blue
fOOd dye O I O O I O O O O O O O I O O I O I O O O O O O O
4. Cross section of a dyed apple lenticel, or an open
lenticel . C O O O O C O O O O O O O O C O O O O O O O O O
5. Structure of a non-dyed apple lenticel, or a closed
lenticel (C.S.) without aperture at the cuticle . . . . .
6. Structure of a non—dyed apple lenticle or a closed
lenticel (C.S.) with aperture at the cuticle . . . . . . .
7. Structure of a dyed apple lenticel, or an open
lenticel (C.S.) O O O O O O O O C O O O O I O O O O O O C
8. The relationship of measured total Ca content to the
estimated increase in Ca content determined on the basis
of weight gain for 'Jonathan' apple fruit hydrocooled in
solution containing 2% CaCl2 plus 0.1% L-77, a surfactant.
9. Relationship between ATIn and solution infiltration
measured as weight gain of 'Jonathan' apple fruit re-
sulted from hydrocooling . . . . . . . . . . . . . . . . .
10. The relationship between the estimated maximum AV
and solution infiltration measured as weight gain
of 'Jonathan' apple fruit resulted form hydrocooling . . .
11. The relationship between Jc and B1 . . . . . . . . . . . .
12. A typical cooling curve of 'Jonathan' apple fruit . . . .

vi

Page

16

21

21

23

23

25

25

35

42

44
61

64

Appendix

Appendix

Appendix

Appendix

Appendix

II.

III.

IV.

LIST OF APPENDICES

The estimation of the increase in fruit Ca based
on weight gain of apple fruit submerged in a sol-

ution of known CaCl2 content . . . . . . . . . . .

The effect of 55 torr partial vacuum and 21°C
temperature drop on AV . . . . . . . . . . . . . .

The relationship between ATm and AV of 'Jonathan'
apple fruit during cooling . . . . . . . . . . . .

The derivation of mass average temperature from
the temperature at the center of apple fruit . . .

The estimation of AV from ATm . . . . . . . . . .

vii

Page

55

S6

58

60

67

INTRODUCTION

The useful life of a mature but unripe apple fruit under Optimal
storage conditions is limited by how slow it progresses through natur-
ally occurring senescence processes, decay caused by pathological
agents and its susceptibility to physiological disorders.

Low levels of calcium (Ca) in the fruit are implicated in a number
of physiological disorders during storage such as bitter pit, cork spot
and Jonathan spot. In addition, water core, internal breakdown, low
temperature breakdown, lenticel spot, scald and rot may be intensified
by sub-optimal levels of Ca (3). A more fundamental role of Ca in de—
laying the senescence of apple fruit is evident by the faster post-
climacteric rate of respiration of fruit with low Ca content (1), the
more rapid loss of membrane integrity of pit-prone fruits (2, 22), and
the lower permeability of fruit tissue low in Ca (39). The obvious
solution to the problem of Ca deficiency is to increase the Ca content
of the fruit to the extent that it will eliminate or considerably mini-
mize the occurrence of these Ca-deficiency related disorders.

Considerably effort has been devoted to the development of methods
to increase the Ca content of the fruit, yet none of the current methods
appears adequate. There is great need for the development of an eco-
nomical and reliable means to increase the Ca content of the fruit suf-

ficiently to retard the development of disorders.

LITERATURE REVIEW

Most Ca is deposited in apple fruit during the first 4 to 6 weeks
of growth and development following anthesis. Subsequently, little ca
moves from the vegetative part of the tree to the fruit, but some Ca
may move from the fruit to the leaves and shoots under certain condi-
tions (48). Young fruits, because of a relatively large surface area
and a highly permeable cuticle, have a high rate of transpiration. Ca
absorbed by the roots is transported to the fruit via xylem and is rel-
atively mobile. With increasing fruit size, transpiration diminishes.
Assimilates are transported via the phloem, in which Ca is not mobile
(35). Two possible means of increasing the Ca content of the fruit are
to decrease the leaf/fruit ratio subsequent to the cessation of the in-
flux of Ca to the young fruit and to supply Ca ion directly to the fruit.

It has been known for many years that severe pruning in the dor—
mant period increases the likelihood of bitter pit in the subsequent
apple crop (9). It was concluded that the increase in tree vigor due
to severe pruning resulted in a high leaf/fruit ratio and, hence, more
competition from the leaves for water and nutrients. Summer pruning re-
duces the leaf/fruit ratio and minimizes the problem associated with
severe pruning in the dormant period (32). The fruit Ca is reported to
be increased by approximately 1 mg/100 g fresh weight when summer
pruning is employed (27). This increase from summer pruning, however,
does not provide an adequate increase in Ca content of the fruit to

2

3
overcome the storage disorder problems. Furthermore, the labor cost
prohibits its adoption by growers in the U.S.A.

Attempts have been made to increase the Ca content of the fruit by
spraying the trees with Ca salt solutions during the growing season.
Prebloom calcium chloride or lime sprays on 'Spartan' apple trees did
not increase fruit Ca level or decrease breakdown incidence (20), where-
as, postbloom Ca salt sprays have been helpful. In general, their ef-
fectiveness when applied after fruit set increases with increasing con-
centration of the Ca salt and frequency of spraying. The highest use
able concentration is limited by the level which damages the leaves or

fruits (44). Four or more sprays of 0.6% (W/V) CaCl to the tree or a

2
single spray of 2 or 4% (W/V) CaCl2 just before harvest seems to be
similarly effective for increasing the Ca content of the fruit by about
40 ppm dry weight or more (31). Ca chloride and Ca nitrate have proven
to be more effective than Ca lactate or Ca acetate (46). Ca phosphate
has proven ineffective as a tree spray (23).

A postharvest dip or drench of apple fruit with 4% (W/V) CaCl so-

2
lution, the most widely used and most effective method to remedy the Ca-
deficiency related disorders, generally increases the Ca content by at
least 80 ppm (31). In general, Ca uptake by the fruit increases with
increasing concentration of the Ca salt, with the highest concentration
being limited by the level at which an unacceptable amount of damage
occurs to the treated fruits. CaCl2 is the most effective among the Ca
salts. The addition of a wetting agent alone to the dip solution may
reduce the amount of CaCl2 retained at the surface of the fruit (31),

and hence, reduce the amount of Ca uptake (24). Modifying the viscos-

ity of the dip solution by thickener, Kelzan (Keltrol) or arrowroot,

4

greatly increased the Ca uptake by the fruit, with up to 825 ppm dry
weight increase being reported (24, 8). The thickeners apparently
caused the adherence of a greater volume of the dip solution to the
fruit surface, favoring Ca penetration into the fruit (19). Combining
a thickener and a wetting agent resulted in greater uptake of Ca by the
fruit (24) with a higher rate of uptake (8) than with either alone.
Unfortunately, the solution is difficult to prepare in large quantity
since vigorous stirring is required to make the gel-like solution.
Spillage of this slippery solution can be a safety hazard to the workers.

Lecithin, a phospholipid used as a general food additive, added in
either a Ca(NO3)2 or a CaCl2 dip solution reduced the incidence of bit-
ter pit and enhanced the Ca uptake by the fruit (8, 37). It is postu-
lated that lecithin, a highly polar compound with a lipophilic fatty
acid "backbone" and hydrophilic choline "head", might assist the move-
ment of Ca ion through the waxy cuticle of apple fruit (37). Accord-
ingly, three Ca ion-containing lipophilic compounds which may penetrate
through the hydrOphobic cuticle were synthesized and applied to 'Golden
Delicious' apples (38). In the short run, the compounds enchanged the
rate of Ca uptake, but after prolonged storage, none of these compounds
was more effective than CaCl2 in reducing fruit softening. The residue
of lecithin on the surface of the fruit at the end of the storage per-
iod must be removed, and this washing is an additional cost. Further-
more, lecithin is a rather expensive chemical.

It was observed that the dip solution was absorbed by 'Jonathan'
apple fruit grown in Australia, when the temperature of the dip solu-
tion was lower than that of the fruit (42). The amount of dip solution

absorbed increased with decreasing temperature of the dip solution and

5
increasing duration of dipping. The solution entered the fruit through
the open calyx, typical of the cultivar employed, and moved into the
core cavity. There was no advantage in respect to controlling break-
down by using a 5°C dip solution instead of a 20°C dip solution for
20°C fruits. A similar treatment applied to 'Spartan' apples (19) re-
vealed that 38°C fruit dipped in 0°C solution resulted in an increase
of 53 ppm Ca in the fruit flesh.

Applying a vacuum to the surface of the CaCl solution in which

2
apples are immersed results in air being forced out of the fruits, so
that upon returning to normal pressure, a small amount of solution
enters into the fruits. Vacuum infiltration (225 mm Hg for 2 min) with
solution up to 4% (W/V) CaCl2 has increased the mean Ca content in
'James Grieve' apple from 3.3 to 15 mg/100 g fresh weight. Fruits thus
treated were found (43) to be free of bitter pit and breakdown, more
firm, and greener in color after 12 weeks of storage in air at 3.5°C
than untreated fruits. New Zealand 'Cox's Orange Pippin' and Australian
'Granny Smith' apples responded extremely well to the vacuum infiltra-
tion methods (43).

Positive pressure has proven to be effective in forcing a Ca salt
solution into the fruit. The amount of Ca uptake can be controlled by
the Ca concentration in the solution, the amount of pressure and the
duration of treatment (30). A 518.2 to 1036.4 mm Hg positive pressure
has increased the fresh weight of the fruit by l to 4%. Pressure in-
filtrated 'Golden Delicious' apples had no loss in firmness after pro-
longed storage at 0°C. This effect of Ca on firmness was obtained re-
gardless of whether the fruits were treated soon after harvest or after

three months of storage (29). Both the vacuum and pressure infiltration

6
methods, if performed on an commercial scale, would require large,
strongly constructed metal chambers that would be costly to build and

time consuming to operate.

OBJECTIVES OF THE STUDY

Results of the pressure and vacuum infiltration methods suggest
that the migration of solution in the the intercellular spaces (ICS) of
a submerged apple fruit can be induced, provided a positive pressure is
established between the ambient atmosphere and the internal atmosphere
of the ICS of the fruit cortex. \

Another possible method of establishing a favorable pressure rela-
tionship is by temperature differential. A warm fruit submerged in a
cold solution used as a cooling medium could provide a pressure differ-
ential and may cause solution infiltration. Previous studies (19, 42)
indicated that the submersion of warm fruit in a cold CaCl2 solution
did not yield more effective control of fruit breakdown than the con-
ventional dip or drench method. The ineffectiveness was attributed to
either the uptake of solution into seed cavities through Open calyx or
to the insufficient increase in fruit Ca content, which could be the
result of insufficient amount of solution infiltration into the fruit
cortex.

Assuming that the solution infiltrated into the fruit via some of
the open lenticels at the surface of the fruit, the amount of solution
infiltrated could be increased by either increasing the temperature
differential or reducing the threshold pressure required to initiate
solution infiltration. There are limitations on the magnitude of the

temperature differential that can be safely induced by hydrocooling the

7

8

fruit. The threshold pressure required for solution infiltration
through small apertures depends on many factors, one of which is the
surface tension of the solution (41). The lower the surface tension,
which can be reduced by the addition of a surfactant, the lower the
threshold pressure.

It was the purpose of this study to investigate the effects of
surfactants and temperature differentials on solution infiltration in‘
'Jonathan' apple fruit, and to assess these treatments in increasing

the Ca content of the fruit for prolonging their storage life.

GENERAL MATERIALS AND METHODS

The 'Jonathan' apple fruit used came from three lots obtained from
two sources. Lot A was harvested Sept. 23rd, 1977 at the Horticulture
Research Center, East Lansing. Lot B and C were purchased on Dec. 16th,
1977 and Feb. 3rd, 1978, respectively, from the Rasch Brothers Orchards
near Sparta, Michigan. The latter were harvested on Sept. 23rd and
24th, 1977 and stored CA without postharvest calcium treatment.

The fruit was stored at -0.5°C in one bushel wooden crates over-
wrapped with 1.5 mil polyethylene bag to maintain high relative humid-
ity. The fruits were sorted and randomized within the lot before stor-
age so that subsequent handling would be minimized.

The fruits for experiments were sorted to remove those with vis-
ible surface defects of a physiological, pathological or mechanical
nature. Each fruit was selected for weight (85 - 145 g) and diameter
(64 - 70 mm). The pedicle, if present, of the fruit was trimmed to a
length of approximately 6 mm to minimize weight gain caused by hydra-
tion of dry pedicle. All loose particles and debris on the surface of
the fruit were removed with puffs of air.

The selected and prepared fruits were held in a room or growth
chamber at the desired experimental temperature for 24 to 36 hours
prior to the treatment. It was found that 24 hours was necessary for
the temperature at the center of the fruit to equilibrate to the tem-
perature of the room or growth chamber. The temperatures of the solu-
tion were conditioned the same manner as that of the fruits.

9

10
All experimental solutions were prepared with distilled water con-

taining either 2 or 4% (W/V) CaCl.lJ. Blue food colorgl, when used was

2
added at a rate of 0.1% (V/V). The surfactants, x9772/ and L-77fll, emr
played in some experiments, were added at a rate of 0.1%, which is ten-
fold the critical micelle concn of both surfactants (6, 11).

The submersion treatments utilized 5 liters of treatment solution
contained in an 8 liter bucket kept in a room at the temperature of the
solution. Ten fruits were placed into the bucket and kept submerged
just below the surface of the solution with a piece of perforated plas-
tic disc for a selected period of time. This simple arrangement en-
sured that fruits of different replicates within a treatment received
the same amount of cooling. Upon removal from the solution, the fruits

were rinsed in.tap water and blotted with paper towels, with particular

attention to drying of the stem and calyx ends of the fruits. The

fruits were then placed on their sides in a 21 i 1°C room for 20 min, then

then turned over and held for another 20 min, to ensure complete evap-
oration of water adsorbed on the surface of the fruit before weight
measurements were taken.

The weight of each apple was measured before and after submersion
treatment with a Mettler 160 tap loading balance to the nearest mg.
The weight change was expressed as either mg/fruit or mg/100 g fresh

weight 0

 

CaCl anhydrous with purity of 96.3%. J. T. Baker Chemical Co.
PhilIipsburt, N.J.

4% dye content, Seeley-Morris Extract Co. Detroit, Mich.
Chevron Chem. Co. Ortho Division.

Union Carbide Corp.

ll

Tissue blocks, 2 x 2 x 2 mm3 in size, containing lenticels were
excised from apple fruits and fixed in FFA solution (15). Dehydrated
in serial tertiary butyl alcohol - ethyl alcohol solutions and embedded
in paraffin (15). Serial sections of the embedded tissue were cut with
a rotatory microtome at 12 um thickness. Sections were mounted,
stained with fast green and counter-stained with Sudan VI.

Freshly excised tissue blocks containing lenticels were mounted
and frozen in Optimum-Temperature Compoundl/ on metal stubs (33). Sec-
tions 32 pm in thickness were cut in a cryostat at ~22°C and placed on
glass slides. A drop of OTC was placed over the section on the glass
slides and covered with cover-slip. The edges of the cover-slip were
sealed with melton wax. Cryostat sections of apple fruit thus mounted
could be preserved for two weeks.

The eray intensities of Ca and C1 in apple fruit tissue were
measured with an electron microprobegj operated at 15 Rev accelerating
voltage, 0.02 “A sample current at 500x magnification. For semi-
quantification of the elements (34), the electron beam was set to scan
horizontally at l msec/200 pm and vertically at 80 msec/160 um over an
area 160 x 200 um2 on the tissue surface approximately 2 mm beneath the
cuticle. The X—ray intensities of Ca (Ka’ 3.359 A) and Cl (Ka’ 4.728 A)
were counted for 10 sec and repeated 10 times on the same area.

The calcium content was determined by atomic absorption spectro-
photometry for whole fruit of known weight with seeds removed. The

tissue was macerated in 100 ml of deionized distilled water in a Wareing

 

y
y

OTC, -15 to -30°C, Fisher Scientific Co.

ARL, Mbdel EMXPSM

12
blender for 3 min. The addition of water gave the macerated tissue a
uniform consistency. An aliquot of approximately 10 g was weighted to
the nearest mg, and transferred to a crucible and air-dryed at 50°C for
12 hr prior to being ashed at 550°C for 10 hr. The ash was dissolved
in 5 m1 of 0.5 N HCL with 1% LaCl3 (49). The ash solution was analyzed
with an atomic absorption spectrophotometerll. The concn of Ca in ppm
in the ash solution was computed from a standard curve constructed with
solutions of known concn of Ca. The Ca content of the fruit was then
calculated accordingly and expressed as mg Ca/lOO g fresh weight.

The temperature at the center of the apple fruit was measured
through a hole, 6.4 mm in diameter, bored perpendicularly to the stemp
calyx axis from the equator to the center of the fruit. A thermometer,
with resolution to 0.1°F, was inserted and the opening was sealed with
non-phytotoxic molding rubber to prevent the seepage of cooling solu—
tion into the fruit. The fruit containing the thermometer was lowered
into the cooling solution so that the whole fruit was submerged with
the fruit-thermometer junction at the surface of the solution. The
temperature readings were taken at two—minute intervals for forty

minutes, starting immediately after the fruit was completely submerged.

 

AJ Beckman, Model DB-G grading spectrophotometer equipped with Model

1501 atomic absorption accessary with laminar flow burner assembly.

EXPERIMENT I. FRUIT WEIGHT GAIN DURING HYDROCOOLING

Introduction: The intercellular spaces (ICS) of apple fruit flesh oc-

 

cupy up to 25% of the total volume of the fruit at maturity (36). The
major portion of the ICS is filled with gas (14). In a study of apple
fruit porosity, nitrogen gas under 40 cm water column pressure intro-
duced to the center of the fruit flowed through the fruit with ease via
the interconnecting portion of the ICS and exhausted to the external
atmosphere, presumably, via open lenticels (14). A temperature reduc-
tion of the fruit lowers the pressure in the ICS. When totally sub-
merged in an aqueous solution, the ambient pressure could force the ex-
ternal solution into the ICS, and if so, it could be measured as a
weight gain of the fruit. The purpose of this experiment was to study
the possible solution infiltration into the totally submerged fruit
during hydrocooling by means of fruit weight gain and occurrence of
blue coloration in the fruit flesh.

Materials amd Methods: Variables considered were solutions either with
or without blue food dye, fruits at either 22.5 or -0.5°C, and solu-
tions at either 22.5 or -0.5°C, for a total of eight treatment combina-
tions. Thirty fruits were subjected to each treatment for a period of

one hour. All solutions contained 4% (W/V) CaCl Weight gain was

2.
measured for individual fruit as mg/fruit. Since the standard devia-
tions of the treatments were nearly proportional to their means, data
were transformed by log10 and subjected to onedway analysis of variance

(21).

13

14
Results: The presence or absence of food dye in the cooling solution
was not significantly related to weight gain of the fruit, whereas the
solution temperature and fruit temperature contributed significantly to
weight gain. The highly significant interaction of solution tempera-

ture and fruit temperature (Table 1) indicated their effect on weight

Table 1. Statistical evaluation of the effects of food dye (FD), solu-
tion temperature (ST) and fruit temperature (FT) on weight

gain of 'Jonathan' apple fruits.

 

 

Source of variation d.f. F

total 239

treatment 7 59.6**
FD 1 0.01
ST 1 93.2**
FT 1 210.5**
FD X ST 1 0.1
FD X FT 1 5.6
ST X FT 1 107.7**
FD X ST X FT 1 0.2

error 232

 

**
significant at a = 0.01

gain to be interdependent. The submersion of warm fruit (22.5°C) in
cold solution (-0.5°C) yielded a substantial increase in weight of the
fruits over the other treatments (Table 2). The presence of blue col-
oration in the cortex, which could be observed through the cuticle
(Figure 1), occurred only to fruits with substantial weight gain. It
was indicative that the weight gain was a result of the infiltration of
the cooling solution into the fruit.

Based on the assumption that weight gain is due, at least partial-

ly, to the infiltration of solution, any dissolved chemicals in the

Figure l.

15

The surface of 3 'Jonathan' apple fruit hydrocooled in 4%

CaClz solution tinted with blue food dye. Note the blue

color around the lenticel. 20X.

 

 

16

 

17
Table 2. The effect of solution temperature (ST) and fruit temperature
(FT) on mean weight gain of 'Jonathan' apple fruit submerged

for one hour.

 

 

 

Treatment Weight gain
ST (°C) FT (°C) (mg/fruit)
"°°5 -0-5 18.1 2.5g
-0.5 22.5 328.3 27.1
22.5 -0.5 17.7 1.6
22.5 22.5 27.2 2.6
1/

- S. E. based on 60 replications

solution should enter the fruit with the solution unless there is some
mechanism to exclude the entry of the chemicals, but not water and dye.
If weight gain results entirely from solution infiltration, 328 mg/

fruit weight gain from 4% CaCl aqueous solution would be equivalent to

2
an increase of 3.69 mg Ca/lOO g fresh weight for a fruit weighing 120 g
(Appendix I). When this amount is added to the average native Ca con-
tent, 1.49 mg Ca/lOO g fresh weight (Exp. VI), the total Ca content of
the fruit would approach the 5.5 mg Ca/lOO g fresh weight that is con-
sidered adequate for Ca to produce a beneficial effect on apples during

long-term storage (28). Since a one-hour submersion period is imprac-

tical for commercial use, a quicker means of Ca infiltration is needed.

EXPERIMENT II. THE DISTRIBUTION OF DYE SOLUTION IN APPLE CORTEX
AND THE ANATOMICAL DIFFERENCES BETWEEN OPEN AND CLOSED LENTICELS

Introduction: Areas of blue coloration were observed beneath the cuti-
cle of warm fruit (22.5°C) which exhibited a substantial amount of
weight gain following submersion in cold (-0.5°C) dye solution (Exp.
1). An examination of the distribution of dye solution in the fruit
cortex was made to provide clues for the cause of fruit weight gain
during hydrocooling. Since the lenticel offers a possible portal of
entry for the solution, its structure was carefully observed.

Materials and Methods: The flesh of apple fruits showing blue colora-
tion were examined visually and with the aid of a dissecting micro-
scope. Tissue blocks containing a lenticel surrounded by a small
amount of dye, shown as a dyed lenticel in Figure 1, and containing a
lenticel without dye (non-dye lenticel) excised from the same fruit
which had been submerged briefly in 4% CaCl2 solution with 0.1% blue
food color were processed by the paraffin method for anatomical exam!
ination. Tissue blocks with dyed and non-dyed lenticels were also sec-
tioned with a cryostat and examined.

Results: The blue coloration always appeared in the tissue adjacent to
some of the lenticels on the fruits submerged in cold dye solution. In
cross section, this coloration was located at the periphery beneath the
cuticle in isolated areas (Figure 2). The mean depth of penetration

was 3 mm, with a maximum depth of 7 mm. No coloration was observed in

18

19
the cortex near the pedicle or calyx, nor in the seed cavity. Em?
ploying the dissecting microscope, the blue color was seen clearly
within the intercellular spaces (Figure 3). Sections of dyed lenticels
prepared by the cryostat showed that the dye was distributed as dis-
crete blue dots beneath the lenticels (Figure 4).

Serial sections of the non-dyed lenticels revealed that some had
no aperture at the cuticle surface (Figure 5), others had aperture,
but the surrounding cells were tightly packed without an open space be-
tween them (Figure 6). Serial sections of the dyed lenticels show that
all had apertures at the cuticle and the surrounding cells were loosely
arranged with open spaces between them (Figure 7). Some of these open
spaces connected the cuticle aperture and the intercellular spaces
(Figure 7).

The presence of coloration immediately adjacent to lenticels sug-
gested that these lenticels were the portals of entry for the solution.
The anatomical examinations of the open lenticels with dye penetration
confirmed that their structure would permit the entry of solution under
pressure into the cortex of the fruit. The discrete blue dots in the
cryostat section of open lenticels strongly suggested that there were
specific routes through which the solution migrated to the cortical
tissue. Since the cortex is composed of thindwalled parenchyma cells
and randomly distributed intercellular spaces, the logical route is the
intercellular spaces. This is substantiated by the presence of blue
color in the intercellular spaces at the edge of the dye-colored area
where individual sections of the intercellular spaces containing blue

color were discerned.

Figure 2.

Figure 3.

Cross section of a 'Jonathan' apple fruit hydrocooled in 4%

CaCl2 solution tinted with blue food dye. Note the blue col-

oration at the periphery of the fruit immediately beneath the

cuticle.

Cross section of cortical tissue of 'Jonathan' apple fruit

hydrocooled in 4% CaCl solution tinted with blue food dye.

2

Note the irregular-shaped blue coloration between the par-

enchuma cells, where the intercellular spaces are located.

120x.

 

 

21

 

22

Figure 4. Cross section of a dyed apple lenticel, or an open lenticel,
prepared by the cryostat. Note the dye is distributed as

discrete blue dots as indicated by arrows. 2,444X.

Figure 5. Structure of a non-dyed apple lenticel, or a closed lenticel

(C.S.). Note that there is no aperture at the cuticle. 2,444X.

 

23

 

24

Figure 6. Structure of a non-dyed apple lenticel, or a closed lenticel
(C.S.). Note the tightly packed cells around the aperture at

the cuticle. 1,000X.

Figure 7. Structure of a dyed apple lenticel, or an open lenticel (C.S.).
Note the loosely arranged cells around the aperture at the

CUtiCleo 2,444X.

 

25

 

EXPERIMENT III. DYE AS A TRACER FOR CaCl2 IN APPLE FRUIT TISSUE

Introduction: It was found that warm intact apple fruit submerged in a

 

cold 4% CaCl solution tinted with blue food dye gained weight substan-

2
tially (Table 2) and had blue coloration in the cortical tissue. The
dye in the solution was shown to be incidental and not causal to fruit
weight gain (Table l). The presence of this blue coloration in the
cortical tissue in fruits having weight gain suggests solution infil-
tration into the cortical tissue. The CaClZ, as a solute, likely
entered into the cortical tissue as well. An experiment was conducted
to determine whether CaCl2 entered the cortical tissue and, if so, to
relate its presence to the distribution of the dye in the fruit corti-
cal tissue.

Materials and Methods: Three warm (21°C) fruit were submerged in cold

(-1°C) 0.1% blue food dye solution with or without 4% CaCl for a per-

2

iod of one hour. Tissue blocks, 3 x 3 x 3 mm3 in size, with cuticle
were excised from both colored and non-colored regions of the same
fruit. After freeze drying (40), they were mounted on carbon discs

1/

with Television Tube-Koat- and coated with a thin layer of carbon in a
vacuum evaporatorgl, a method of tissue preparation that prevents re-
distribution of water soluble compounds in the tissue. Both calcium

and chlorine were semi-quantified with a microprobe.

 

_1_/

'21 Varian, Mbdel VE 10.

Co E o EleCtrons o

26

27
Results: The Xpray intensities of the elements obtained from plant
tissue prepared as outlined above can be positively correlated but can-
not be transformed to determine the concentration of the elements in
the tissue. Comparisons of eray intensities, therefore, were limited
to the same element in tissue of different treatments. The presence of
blue dye in the tissue did not affect the Ca and Cl content in the

tissue. The presence of CaCl in the treatment solution yielded a tre-

2
mendous increase of Ca and C1 in the tissue; furthermore, the increase

occurred only in tissue with blue coloration (Table 3).

Table 3. The mean X-ray intensity of calcium and chlorine in cortical
tissue of 'Jonathan' apple fruit hydrocooled in dye solution

either with or without CaClz.

 

X-ray intensity

 

 

Coloration of CaCl in Calcium Chlorine
cortical tissue soluéion (counts per 10 sec.)
1/
+ + 443.2 56.3— 344.7 58.6
— + 19.7 0.6 1.5 0.4
+ "' 31.2 3.4 1.8 007
- -' 33.7 709 2.1 005
y

S.E. of 3 replications.

There is no doubt but that Ca had entered the cortical tissue of
the fruit. Its presence was indicative that hydrocooling of apple

fruit with solution containing CaCl could increase the Ca content of

2

the fruit. The dye proven to be a good tracer of CaCl at the termina-

2
tion of the submersion treatment. The distribution of blue coloration
in apple flesh described in Exp. II was similar for calcium. Since Ca

applied to the fruit surface was found (47) to penetrate the cuticle

28
and migrate into the core of the fruit, it is likely that Ca once in

the intercellular spaces would move throughout the fruit.

EXPERIMENT IV. THE EFFECT OF SURFACTANTS ON FRUIT WEIGHT GAIN

Introduction: A possible means for increasing the rate of solution in-

 

filtration and thereby shortening the duration of submersion is to
lower the surface tension of the solution by adding a surfactant. It
has been demonstrated that mass solution infiltration through open
stomata of leaf can be induced by adding a small amount of surfactant
to the solution (11). The opening of the ICS at the open lenticels,
which may serve as portals of entry for solution infiltration, have
similar dimensions to stomata. Since X-77 and L-77 can lower the sur-
face tension of water to 33 and 24 Newton respectively (6, 11). These
two surfactants were tested to study their effect on fruit weight gain
which could be resulted from solution infiltration induced by hydro-
cooling.

Materials and Methods: The weight gains of 50 fruits with a 21°C ini-

tial temperature submerged in a 4% CaCl plus 0.1% X977 aqueous solu-

2
tion at -l°C for 10, 20, 30,45 and 60 min were measured. Similarly,

weight gains of 50 fruits at 21°C were obtained in 4% CaCl plus 0.1%

2
L-77 aqueous solution at -1°C for 5, 10, 15 and 20 min. Weight gains
of 40 fruit at 21°C were obtained in 4% CaCl2 aqueous solution at -1°C
for 15, 30, 45 and 60 min as controls.

Results: A 20-min treatment with X—77 gave a weight gain equivalent to
60-min submersion in solution without the surfactant. L-77 was more

effective than X977, producing in 20 min more than triple the weight

29

30
gain in 60 min for the control (Table 4). It is evident that the sur-

factants markedly enhanced fruit weight gain. Since one of the effects

Table 4. The effect of surfactants on mean weight gain of 21°C

'Jonathan' apple fruit during submersion in 4% CaCl solu-

 

 

 

2
tion at 91°C.
Submersion Mean weight gain (mg/fruit)
duration (min) control X977 (0.1%) L-77 (0.1%)
5 - - 164.8 13.6y
10 - 79.6 104 423.5 3205
45 202.9 31.5 718.2 43.8 -
60 283.9 29.8 1010.7 79.5 -
_1_/

S.E. of 50 replications.

of surfactant is to enhance solution infiltration through small apper-

tures, this result supported the hypothesis that fruit weight gain was

resulted from solution infiltration. With the aid of L977, an adequate
amount of Ca, 5.71 mg/fruit, could be infiltrated into the fruit in

less than 10 min.

EXPERIMENT V. THE EFFECT OF SUBMERSION DURATION AND INITIAL
FRUIT TEMPERATURE ON FRUIT WEIGHT GAIN

Introduction: The weight gain of warm fruit submerged in cold solution
was shown to be, most likely, due to the infiltration of solution into
the intercellular spaces. As the fruit is cooled, the volume of gas
within the intercellular spaces is reduced, which results in a lower
pressure in the intercellular spaces as compared to the ambient atmos-
phere pressure. It is assumed that this pressure differential is the
driving force for solution infiltration. According to the gas laws
this pressure differential is proportional to the temperature drop (AT),
which is the difference in temperature of the fruit before and after
cooling. With AT being a function of both the initial fruit tempera-
ture (To) and the cooling, or submersion duration (t), an experiment
was designed to study the effect of these two factors on fruit weight
gain at a constant cooling medium temperature (T1).

The hydrocooling machinery employed in the cooling of fresh pro-
duce is designed to maintain the temperature of cooling water at ap-
proximately 5°C, the temperature used in this experiment. The range of
initial temperature of the fruit chosen for this experiment was 10 to
20°C, as the approximate range of the temperature for 'Jonathan' apples
harvested in Michigan.

Materials and Methods: The weight gain of fruits initially at 10, 15

and 20°C when submerged in 2% CaCl plus 0.1% L-77 solution that was

2
31

32
refrigerated to maintain 5 i 0.5°C for 10, 20 and 30 min were measured.
Fifty fruits were used in each treatment.
Results: The fruit weight gains increased significantly with both in-
creasing submersion duration and with higher initial fruit temperature

(Table 5). Since it was proposed that the amount of solution infil-

Table 5. The mean weight gain of 'Jonathan' apple fruit under simu-
lated hydrocooling conditions (solution temperature: 5 t

0.5°C, 2% CaCl plus 0.1% L-77).

2

 

initial fruit submersion period
temperature 10 min 20 mdn 30 min
mean weight gain (mg/100 g fr. wt.)

 

10°C 53.6 4.8l/ 147.2 10.9 282.0 18.5
15°c 230.0 15.9 452.6 27.4 595.4 30.7
20°C 367.1 29.3 808.2 52.7 938.4 56.8

 

-£/ S.E. of 50 replications.

trated could be affected by both To and t at a constant T1, the results
suggested that fruit weight gain was a reflection of solution infiltra-
tion. Furthermore, these data could serve as a guide for determining
the required submersion duration for fruits of various initials tem-
peratures in order to achieve a desired amount of weight gain, and
hence, increase in Ca content if the total amount of solution infil-

trated could be measured as fruit weight gain.

EXPERIMENT VI. THE RELATIONSHIP BETWEEN FRUIT WEIGHT GAIN
AND Ca CONTENT IN THE FRUIT

Introduction: It was established in Exp. III. that Ca entered the

 

fruit cortex with the solution, yet the amount of Ca entering the fruit
relative to the weight gain was not determined. The relationship is
herein studied together with an investigation of the validity of the
estimation for the increase in Ca content of apple fruit based on
weight gain, which is assumed to be the result of solution infiltra-

tion, in known concn of CaCl , as estimated in Appendix I.

2
Materials and Methods: Weight gains were measured for 30 fruits at a

 

temperature of 21°C submerged in a solution of 2% CaCl plus 0.1% L977

2
at -1°C for 10, 20 and 30 min. Fruits with weight gains ranging from
approximately 220 to 2300 mg/100 g fresh weight were chosen at an in-
crement of approximately 400 mg/100 g fresh weight, each in duplicate.
The Ca content of these fruits was determined by atomic absorption
spectrOphotometry, and linearly correlated with the estimated increases
in Ca content based on weight gains. The Ca contents of 10 untreated
fruits were also measured.

Results: The linear correlation of measured and estimated increases in
Ca content for fruits with various amounts of weight gains was nearly
perfect, r = 0.9976 (Figure 8). The slope of the regression equation
was 1.04 with a 99% confidence interval of 1.10 to 0.98, which included

the value of 1.00. It is concluded that for each unit of increase in

33

34

Figure 8. The relationship of measured total Ca content to the esti-
mated increase in Ca content determined on the basis of
weight gain for 'Jonathan' apple fruit hydrocooled in a solu-

tion containing 2% CaCl2 plus 0.1% L-77, a surfactant.

35

Y =1.45+1.04 X
r = 0.9976

MEASURED Ca CONTENT (mg/1009 FR. WT.)

 

O 4 8 12 16
ESTIMATED Ca INCREASE (mg/1009 FR. WT.)

36
estimated Ca, there is the same unit of increase in the measured Ca in
the fruit. The intercept of the regression equation was 1.45 with an
estimated S.E. of 0.014, which was not significantly different from the
Ca content, 1.49 t 0.042 mg Ca/lOO g fresh weight, of the untreated
fruits.

The total Ca content of the fruit, according to the regression
equation, is the sum of the native Ca content and the estimated infil-
trated Ca derived from the weight gain. It is obvious that the weight
gain is a true reflection of the weight of the solution infiltrated
into the fruit. Accordingly, the final Ca content of fruit hydrocooled
in a solution containing a known concentration of CaCl2 can be esti-

mated with a reasonable degree of accuracy from the weight gain and the

native Ca content of the fruit.

EXPERIMENT VII. SOLUTION INFILTRATION INTO APPLE FRUIT
INDUCED BY PARTIAL VACUUM

Introduction: The fruits of Exp. 1. had been warmed from 0 to 21°C be-

 

fore they were subjected to the submersion treatments in 90.5°C solu-
tion. There was the possibility that the warming had inflicted certain
changes on the fruit which favored solution infiltration during hydro-
cooling. For example, it has been shown (7) that the number of open
lenticels, which are the portal of entry for solution infiltration, may
increase when fruits are exposed to an environment of low humidity.

The relative humidity in the room in which the fruits of Exp. I. were
warmed was 30 to 35% and 90 to 95% in the storage room.

Infiltration of solution into apple fruit can also be achieved by
partial vacuum (43). From the gas laws it can be calculated that a 55
torr pressure drOp has the same effect as a 21°C temperature drop on
the volume change of an ideal gas (Appendix II). This moderate pres-
sure drop should have little, of any, greater effect on the integrity
of anatomical structure of the fruit than has 21°C temperature drop.

It was the purpose of this experiment to investigate the amount of
solution infiltrated into the fruit before and after being warmed from
0 to 21°C. Since three different lots of apple fruit were used in this
study, it was important also to determine if the apples had the same
property regarding solution infiltration.

Materials and methods: A 559torr pressure drop was applied to fruits

submerged in an aqueous solution of 4% CaCl containing 0.1% X977, a

2
37

38

wetting agent, for 45 min. The partial vacuum was released slowly over
a period of l min followed by 14 min of soaking before the termination
of the treatment. The X977 facilitated the escape of air from the open
lenticels that would ensure subsequent solution infiltration. Thirty
fruits from lot A at 0°C were treated in 0°C solution. Thirty other
fruits of the same lot were warmed to 21°C, then treated in 21°C solu-
tion. Another 30 fruits from each of the three lots were warmed to
21°C and treated in 21°C solution. Weight gains were measured for in-
dividual fruits of all treatments.
Results: The mean weight gain of apple fruits at 0 and 21°C Of 262 i
28.2 and 328 t 36.7 mg/fruit, respectively, were not significantly dif-
ferent. It has been shown that the only portal Of solution entry for
these apples is an open lenticel. Since it has been shown (Exp. VI)
that the weight gain is the sole result of solution infiltration, it is
evident that the status of open lenticels, both the total number of
Open lenticels of a fruit and the degree of opening of individual len-
ticel, was not affected by the temperature change.

The mean weight gain of fruits from the three lots were 328.6 1
36.7, 307.8 1 32.2 and 282.9 i 35.7 mg/fruit. Since they were not sig-
nificantly different from each other, it is indicative that they were

of similar property pertaining to solution infiltration.

EXPERIMENT VIII. THE EFFECT OF FRUIT TEMPERATURE
REDUCTION ON SOLUTION INFILTRATION

Introduction: In previous experiments an increase in weight gain of

 

the fruit was affected by the initial temperature of the fruit and the
length of the submersion duration, both of these factors affect the ex-
tent Of fruit cooling. Weight gain induced by hydrocooling has been
shown to be a result of the amount Of solution infiltrated into the
fruit cortex (Exp. VI). The volume occupied by the infiltrated solu-
tion, AV, is the difference between the structural volume of the ICS
and the volume occupied by the cooled internal atmosphere. The maxi-
mum available AV can be estimated (Appendix V), and is shown (Appendix
IV) to be a linear function of AT, the temperature reduction of the
fruit. This experiment was designed to study the quantitative rela-
tionship between AT and fruit weight gain.

Materials and Methods: The change in temperature during cooling at the

 

center of the fruit was measured by a thermometer sealed into the fruit
under the same treatment condition as weight gain was measured in Exp.
V. Duplicate fruits of 6.4, 6.7 and 7.0 cm in diameter were used in
each cooling treatment. The mass average temperature, Tm, was then
calculated using the information derived from the change of temperature
at the center of the fruit during cooling (Appendix IV). The mean
ATm's Of fruits of each cooling treatment were then correlated with the
corresponding mean weight gains (Table 5).

39

40
Results: The ATm's and weight gains obtained under similar cooling
conditions are almost perfectly correlated in a positive linear fashion,
r I 0.9938 (Figure 9). This provides further support that solution in-
filtration is induced by cooling of the fruit. Furthermore, the amount
of solution infiltrated can be predicted from ATm using the regression
equation. Unfortunately, as shown by Kopelman £3 31. (18), the cooling
of individual apple fruit, thus ATm, cannot be predicted with reason-
able accuracy from the initial temperature of the fruit, cooling medium
temperature, cooling period and the size of the fruit. Otherwise, so-
lution infiltration Of 'Jonathan' apple fruit under various cooling
conditions could be estimated by calculation. Nevertheless, the data
of Table 5 should serve as a useful guide for the practical application

of hydrocooling to enrich fruit with Ca.

Figure 9.

41

The relationship between ATm and solution infiltration

measured as weight gain of 'Jonathan' apple fruit resulted

from hydrocooling. ATm T 9 T ; whereby, Tmo = initial

mo mt

mass average temperature of the fruit and Tmt = mass average

temperature at the end of submersion period t.

42

Y:2.127+0.0108 X
r :O.9938

 

O 200 400 600 800
WEIGHT GAIN [mg/1009 FR. WT.I

EXPERIMENT IX. THE RELATIONSHIP BETWEEN SOLUTION INFILTRATION
AND THE VOLUME CHANGE OF THE GAS WITHIN THE INTERCELLULAR
SPACES OF 'JONATHAN' APPLE FRUIT

Introduction: Anatomical examination (Exp. 11) indicated that solution

 

infiltrated into the cortex of the fruit was located in the intercel-
lular spaces. Since it was shown that the amount of solution infil-
trated was a linear function of ATm (Figure 9) and that the maximum
available AV was proportional to ATIn (Appendix III), weight gain,
therefore, is also a linear function of the maximum available AV. A
remaining question, however, is whether or not the maximum available AV
induced by cooling is large enough to accommodate the volume Of the ob-
served amount of solution infiltrated.

Materials and Methods: The mean ATm's and their corresponding mean
weight gains in previous experiment were employed. The maximum avail-
able AV's were calculated from the ATm's with certain assumptions, as
detailed in Appendix V, and correlated with the mean weight gains in
Table 5.

Results: The linear correlation between the maximum available AV and
weight gain was nearly perfect (r = 0.9938), as shown in Figure 10.

Since the density of 2% CaCl aqueous solution is 1.01 (13), each mg Of

2
the solution should occupy 0.99 ul of volume. The regression coeffi-
cient of the regression equation is 1.06 with a 99% confidence interval
of 1.15 to 0.97, which obviously includes 0.99. In other words, dis-

regarding the value of the intercept, for each 01 of the maximum

43

44

Figure 10. The relationship between the estimated maximum AV and solu-
tion infiltration measured as weight gain of 'Jonathan' apple

fruit resulted from hydrocooling. See Appendix III and V for

definition and determination of AV.

A V [III/1009 FR. WT.I

1200

1000

800

600

400

200

45

Y = 226.6 +1.06 X
r : 0.9938

 

200 400 600 . 800
WEIGHT GAIN [mg/1009 FR. WT.I

46
available induced by cooling, there is a corresponding mg of solution
infiltration. The numerical value Of the AV's in all cases were larger
than that of the weight gains. It is seemingly evident that the maxi-
mum available AV induced by cooling can accommodate the volume of the

Observed amount of solution infiltrated into the fruit.

DISCUSSION

The intercellular spaces (ICS) within the fruit flesh is bounded
by a continuous network of cell walls which can be considered as a
semi-rigid matrix. The available air spaces (AAS) is defined as the
combined volume occupied by the seed cavity and portions of the ICS
that communicate to the ambient atmosphere through Open lenticels. The
gases in the AAS are cooled, when a warm apple fruit is submerged in
a cold solution, the pressure within the AAS decreases. The cold solu-
tion is introduced into the readily accessible AAS when the pressure
difference between the solution and the fruit.AAS exceeds the friction-
al resistance of the open lenticels. This threshold pressure is related
to the surface tension of the cooling solution and decreases as the
surface tension is reduced. Further reduction in fruit temperature is
associated with more infiltrated solution which occupies the volume,
AV, the difference between structural volume of the AAS and the volume
occupied by the cooled internal atmosphere. The fruit temperature re-
duction was measured as AT, the difference between the initial tempera-
ture (To) and the final temperature (Tt) Of the fruit during the cooling
period (t). The amount of solution infiltrated into the fruit was
measured as weight gain, the difference in weight before and after the
fruit was hydrocooled.

Studies (7, 16) of the ontogeny and anatomical structure of apple
lenticels have shown the presence of Open and closed lenticels which

47

48

are indistinguishable to the naked eye. Microsc0pic examinations (7)
revealed that Open lenticel had an aperture at the cuticle as compared
to no aperture for the closed lenticels. There were connecting channels
between Open lenticels and the ICS of the cortical tissue for some of
the open lenticels, which would permit the atmosphere within the ICS to
be in continuum with the ambient atmosphere. This concept of continuum
of atmosphere within and without the fruit is supported by evidence
that a gas introduced under slight pressure into the seed cavity of an
apple fruit was exhausted easily to the external atmosphere by Hoff and
Dostal (14). Additionally, this provided evidence for the assumption
(14) that Open lenticels were the port of gas exhaustion, and to the
conclusion by Burg and Burg (5) that gas exchange in apple fruit oc-
curred through Open lenticels.

It was found in Exp. 1. that in the absence of AT, and thereby no
AP, there was no substantial weight gain, even though the fruit was
submerged in solution for a considerable period. The very small weight
gains recorded for fruits in these treatments could have resulted from
hydration of the fruit tissue itself. The existence of AT when warm
fruit is submerged in cold solution caused the solution to infiltrate
the cortical tissue which resulted in a substantial weight gain. Sim9
ilar Observations were made for 'Jonathan' apple fruit grown in
Australia by Scott and Wills {42); however, contrary to our Michigan
fruit, theirs had Open calyx canals so that the infiltrated solution
moved mostly into the seed cavity.

Exp. II. demonstrated that as the AT was gradually developed, so-
lution infiltration was initiated at the open lenticels where the AAS

is most accessible to the solution. Solution tinted with food dye

49
caused blue coloration around some lenticels, the same phenomenon as
observed by previous researchers (7). Since the AP is developed in a
direction perpendicular to the fruit surface and toward the center of
the fruit, the solution is forced into the ICS of the cortical tissue
immediately beneath the cuticle.

CaCl2 as a solute in the cooling solution entered the fruit in an
amount proportional to the quantity of the infiltrated solution. Con-
sequently, the amount of Ca increase in the fruit could be accurately
estimated based on the weight gain obtained in a solution of known con-
centration of CaClz. It is likely that other solutes and many suspended
particles small enough to be accommodated by the passageways of the
Open lenticels and the ICS would enter the fruit with the solution.

This direct infiltration of solutes into the fruit cortex would pos-
sibly permit the use of lower concentration of chemical in solution em9
ployed for other postharvest treatment of the apple fruit. For example,
diphenylamin (DPA) is employed for the control of superficial scald, a
common storage disorder of apple fruit throughout the world. Presum9
ably, the uptake of DPA into the flesh of the fruit is accomplished by
diffusion of DPA residue on the surface of the fruit across the cuticle.
It was found that the incidence of scald was inversely correlated to
the concentration of DPA in the fruit flesh (12). In order to achieve
an adequate concentration of DPA in the fruit flesh, believed to be 8
ppm (12), 2000 ppm or higher concentration of DPA is used in the drench
or dipping solution. With direct infiltration of DPA solution into the

fruit, the concentration of DPA in the treatment solution could be dra-

matically reduced.

50

Fungicides are sometimes used in the drench or dip solution for
postharvest treatment Of apple fruits to prevent decay caused by cer-
tain fungi (11). If used in the cooling solution, it is expected that
the fungicides be infiltrated into the fruit flesh. Attention should
be paid to the fact that the fungicide residue in the fruit flesh does
not exceed the maximum allowable concentration.

The uptake of Ca, supplied to the fruit as postharvest dip or
drench, into the apple flesh has been proven (4, 47) tO be the result
of diffusion of Ca salt residue on the surface of the fruit across the

cuticle. The superior effectiveness of CaCl in controlling the dis-

2
orders over other Ca salts is attributed by many researchers to its hy-

droscopicity. This physical property allows CaCl to absorb sufficient

2
amount of moisture from the humid storage room and, thus, exist as ions
in solution for easier diffusion. The solution infiltration method
possibly provides a means for reexamining the effect of other Ca salts
in prolonging the storage life of apple fruit.

It was shown (Figure 9) that the amount of solution infiltrated
into the fruit is a linear function of AT. The intercept of the linear
equation, or the lag of the weight gain behind AT could be attributed
to a threshold pressure, occurring as a result of certain characteriS9
tics of the open lenticels in conjunction with the surface tension of
the solution, below which no solution infiltration was possible. Dis-
regarding the intercept, it is apparent that increasing AT is accom-
panied by proportional increases in the amount of solution infiltrated.
When the cooling medium temperature (T1) is maintained constant, in-
creases in initial fruit temperature, To, and submersion duration, t,

cause and increase in AT, and hence increase the weight gain Of the

fruit.

51

Surfactants were employed to reduce the surface tension of the so-
lution and thereby decrease the threshold pressure to be overcome for
initiating solution infiltration through small apertures, as shown by
Schbnherr (41). This served as a means for shortening the submersion
period (t) required to achieve a given amount of solution infiltration.
The use of L977, a surfactant that reduces the surface tension of pure
water to 24 Newton (6), in the cooling solution resulted in a dramatic
enhancement in weight gain (Table 4). The increase in solution infil-
tration achieved in this manner makes the hydrocooling method of post-
harvest Ca treatment for apple fruits a highly feasible possibility for
practical use.

There are several factors that would affect the potential increase
in Ca for apple fruit by the hydrocooling method. One is the concen-
tration of Ca salt in the cooling solution. For a given weight gain,
the Ca content of the fruit may be increased by increasing the concen-
tration of Ca salt in the solution. The actual increase in fruit Ca
was calculated (Appendix I) and verified in Exp. V1. for fruit hydro-
cooled in 2% CaCl solution. It is anticipated that the increase in Ca

2

content for fruit treated in solution with other concentrations of CaCl2
or other Ca salts could be estimated equally well by modification of
the formula used in Appendix I.

It was shown that the increase in Ca content of the fruit was a
function of the amount of solution infiltrated into the fruit, there-
fore, factors affecting solution infiltration would also influence the
increase in Ca content of the fruit.

It is likely that the ICS of an apple fruit consists of some iso-

lated compartments without Opportunity for direct gas exchange with

52
other part of the ICS or open lenticels (14). Meat ICS, however, con-
sists of interconnecting compartments linked to at least one open len-
ticel. The former type of ICS probably does not participate in the
creation of AP, the driving force for solution infiltration, whereas,
the latter type of ICS together with the seed cavity is greatly impor-
tant in the creation of AP. It was derived from the gas laws (Appendix

III) that the maximum AV was proportional to V , the assumed volume of

1

AAS, as long as P and AT/T1 remain constant. Since it was shown (Figure
10) that the amount of infiltrated solution was a linear function of

maximum AV, the size of V Obviously affects the amount of solution in-

l
filtrated into the fruit.

It is postulated that the combined effect of V and the lenticel

1
status, the number of open lenticels per fruit and the extent of indi-
vidual lenticel opening, on solution infiltration could be measured by
the relationship between weight gain and AT in a solution of given sur-
face tension. This relationship deserves further investigation because
it is likely to vary by variety, cultural conditions and other factors
affecting the growth and development of the fruit.

The results of Exp. VI. in which surfactants were added to the
cooling solution indicate that the weight gain is inversely proportion-
al to the surface tension of the solution. This conforms with the
knowledge (41) that lowering of the surface tension reduced the pres-
sure required for initiating solution infiltration through small aper-
tures.

The extent of fruit cooling is important in that weight gain is a

linear function of AT (Figure 9), and furthermore, AT is a function of

To, T1 and t. Weight gain increases with increasing To and t, and with

53

decreasing T Although the AT of any object with regular geometric

1.
shape and known thermal properties can be predicted accurately for any

combination of T,, T and t, the cooling of 'Jonathan' apple fruit can-

1
not be predicted with reasonable accuracy (18). If accurate cooling
could be predicted, the weight gain of apple fruit induced by hydro-
cooling could be predicted for any combination of T., T1 and t in con-
junction with the characterized relationship between weight gain and
AT of a given variety. Nevertheless, various amount of weight gain,

and thus, the amounts of increase in Ca content, can be obtained exper-

imentally for the combinations of T,, T and t, that are of practical

l
usage. From this information, the desired amount of Ca and, possibly,
any other water soluble chemicals could be infiltrated into the fruit
by utilizing the appropriate combination of T0, T1 and t. Further

studies in this area are needed before recommendations to growers can

be made.

CONCLUSION

Solution infiltration into the cortex of 'Jonathan' apple fruit
was induced by cooling the fruit submerged in a solution at lower tem-
perature than the fruit. The solution entered the fruit via open len-
ticels and moved to the intercellular spaces in the cortex at the per-
iphery of the fruit beneath the cuticle. The amount of solution infil-
trated was measured as a weight gain of the fruit.

CaCl2 and, presumably, all other water soluble chemicals, entered
into the fruit with the solution. The amount of increase in fruit Ca
was prOportional to fruit weight gain.

Addition of a surfactant, such as L977, greatly enhanced the rate
of solution infiltration. Increase of fruit Ca up to 16 mg Ca/lOO g
fresh weight was readily achieved with the aid of L977.

The amount of Ca increase of the fruit was affected by initial
fruit temperature, temperature of the cooling solution, submersion dur-
ation, surface tension of the cooling solution, concentration of Ca
salt in the solution and certain morphological characteristics, such as
number of open lenticels per fruit.

A predetermined amount of increase in fruit Ca content could be
achieved by subjecting the fruit to a specific hydrocooling condition.
This hydrocooling method of postharvest Ca treatment for apples is a

highly feasible possibility for practical use.

54

APPENDIX

Appendix I. The estimation of the increase in fruit Ca based on weight
gain for apple fruit submerged in a solution of known

CaCl2 content.

P: purity of CaCl2 in %.

C: concentration of CaCl2 solution in %.

Awt: weight gain of the fruit in mg.
wt: weight of the fruit before treatment in mg.

The prOportion of Ca in CaCl on a weight basis: 40/111 I 0.3604

2
The increase in Ca content in mg Ca/100 g fr. wt.:
(0.3604 - Awt/wt) - 100 0 P o 10.2 0 C 0 10-2
- 3.604 - 10'3 - p . c - Awt/wt
Example:
P I 93.6%. the purity of chemical grade anhydrous CaCl2 used in the
experiments.
C I 4%

Awt I 328 mg
wt I 120 3

mg Ca/lOO g fr. wt. = 3.604 . 10'”3 . 93.6 - 4 - 328/120 I 3.69

55

Appendix II. The effect of 55 mm Hg partial vacuum and 21°C tempera-
ture drop on the AV.

Under constant temperature,
P1V1 I PZV2 (1)

When P1 I 760 mm Hg, P2 = 760 9 55 = 705 mm Hg

substituting these values into Eq. (1),

760 V1 I 705 V2, or Vl

Let AV 8 V2 9 V1 (3)

substituting Eq. (2) into Eq. (3),

= 705/760 V2 (2)

AV I V2 9 705/760 V2

= (l 9 705/760) V2
= 0.07 V2 or AV/V2 = 7%

The volume of gases within the fruit increases as the pressure is
lowered. As gas expands part of it escapes from the fruit through open
lenticels as air bubbles, which comprises 7% of V2. Upon returning to
760 mm Hg, the AV can be replaced with the solution, and the propor-

tionality is retained, i.e., 7% of V can be replaced by the solution.

1

Under constant pressure,
Vl/VZ I T1/T2 (4)

When T1 I 273 + 21 I 294°K, T2 = 273 + 0 = 273°K

substituting these values into Eq. (4),

V1/V2 = 294/273, or V I 273V1/294 (5)

2

Let AV - v1 - v2 (6)

56

57

substituting Eq. (5) into Eq. (6),
AV I V1 9 273 V1/294
I (l 9 273/294) V

1
I 0.07 or AV/V1 I 7%

As the temperature of the fruit is lowered, the volume that the
gas will occupy decreases if the pressure remains constant. This re-
duces the volume by 7% of V1, and represents the maximum amount of
volume the solution may occupy. Accordingly, a 55 mm Hg pressure drop
and a 21°C temperature drop would cause approximately 7% of the volume

of the intercellular spaces of the fruit to be the maximum available

space that may be occupied by the infiltrated solution.

Appendix III. The relationship between AT and AV of 'Jonathan' apple
fruit during cooling. m

The maximum amount of volume (AV) available for solution infiltra-
tion induced by hydrocooling can be calculated as the following:

Under the condition Of constant pressure, the gas law states that:

V2/V1 I T2/Tl (l)
where,

T1 I initial temperature, T2 I final temperature

V1 I volume of gas at T1, V2 I volume of gas at T2

Rearranging Eq. (1),

1 - V2/V1 I 1 9 T /T1, or

2
(V1 - V2)/V1 I (T1 - T2)/T1 (2)
Let AV I V1 9 V2 and AT I T1 9 T2, where T1 > T2

Substituting AV and AT into Eq. (2)
AV/V1 = AT/Tl, or
AV I (Vl/Tl) - AT, or AV I Vl (AT/T1)
The equation states that AV is a linear function of AT.

In the case of these experiments, V is the volume of space in the

l

ICS, which is constant regardless of the temperature of the fruit.

58

Appendix IV. The derivation of mass average temperature from the tem9
perature at the center of apple fruit.

The relationship between mass average temperature, Tm’ and the

temperature at the center of apple fruit, Tc’ is given (17):

Tm - T1 - 1((1‘1 - Tc) («3)
Where, T1 I The temperature of the cooling medium.
3
K Jm/Jc 3/81(sin 81 9 81 cos 81) (b)

Where, Jm I The lag factor for Tm.

Jc I The lag factor for Tc.

81 I The first root of the transcendental function,
NBl I l 9 Bl cot 81, in radian.
and Jc I Ta/(To 9 T1) (c)

Where, Ta I The intercept of the regression equation of log (Tct 9 T1)
vs time t.
To I The initial temperature of the fruit.
Tct I Tc at time t.

Ta is obtained by taking the value of the intercept of the linear re-

gression equation of log (Tc 9 T1) vs t. Jc is calculated by substi-

t
tuting Ta into Eq. (c).
Given (17):
JC I 4(sin B1 9 81 cos Bl)/(281 9 sin 281)
81 is estimated for a given value of Jc from a curve demonstrating the

relationship between Jc and 81 (26) as shown in Figure ll. 81 is

59

60
substituted into Eq. (b) to calculate K, which is then substituted into

Eq. (a) to calculate T .
m

61

Figure 11. The relationship between Jc and 81. A graphical solution

for the equation Jc I 4(sin Bl - 81 cos 81)/(281 9 sin 81)

62

 

 

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63

The Tct's tabulated below are the results for an apple fruit 6.7 cm in

diameter with To 8 67.9°F cooled in a solution at 40.1°F for 40 min.

The Tm's of this fruit are calculated to illustrate the usage of the

equations in the preceding page.

C Tct Tct - T1 t Tct ct ' T1
0 67.9 27.8 22 52.3 12.2
2 67.9 27.8 24 51.1 11.0
4 67.3 27.2 26 50.0 9.9
6 66.1 26.0 28 49.0 8.9
8 64.5 24.4 30 48.2 8.1
10 62.2 22.5 32 47.4 7.3
12 60.6 20.5 34 46.8 6.7
14 58.5 18.4 36 46.2 6.1
16 56.9 16.8 38 45.7 5.6
18 55.1 15.0 40 45.2 5.1
20 53.7 13.6

Tct - T1 vs t is plotted on a semi-log graph paper as show in Figure
12. The regression equation for log (Tct - T1) vs t is resolved for
the straight line portion of the cooling data, i.e., from t = 8 to
t . 32. The intercept of the regression is 37.52 which is Ta.
Substituting Ta = 37.52 into Eq. (c);

Jc = Ta/(To - Tl)

Jc - 37.52/(67.9 - 40.1) - 1.35
Referring to Figure 12, at Jc = 1.35, 81 has the value of 1.76.
Substituting 81 = 1.76 into Eq. (b);

R = 3/Bi (sin 81 - 81 cos 81)

K = 3/1.763 (sin 1.76 - 1.76 cos 1.76) = 0.7226
Substituting K = 0.7226 into Eq. (a);

when t = 10.

Tm . T - K(T1 - Tc), for Tc = 62.2, 1.8., Tet

l
Tm a 4001 " 0.7226(4001 "' 62.2) = 56036
The Tm's and ATm's for all the cooling treatments at solution tempera-

ture (T1) of 40.1°F are tabulated below for reference. All the

64

Figure 12. A typical cooling curve of 'Jonathan' apple fruit.

To - 69.7°F, T = 40.1°F.

l

65

o
w

 

 

50

E...

 

TIME (min)

66
temperatures are given in °F. The ATm's are converted to °C and then
correlated with mean fruit weight gains in Table 5.
Diameter of fruits (cm)

6.4 6.7 7.0

rep 1 rep 2 rep 1 rep 2 rep 1 rep 2 Ave ATm ATm(°C)

t = 10 45.50 46.26 46.75 46.09 46.82 46.07 45.50 4.50 2.50

t = 20 42.92 43.61 44.09 43.63 44.51 44.02 43.05 6.95 3.86

t = 30 41.55 42.18 42.52 42.10 42.92 42.62 41.55 8.45 4.69
To = 59

t = 10 50.07 50.96 51.64 50.82 51.32 51.16 50.44 8.56 4.75

t = 20 45.74 45.85 46.68 47.15 47.16 46.82 46.02 12.98 7.21

t = 30 43.13 43.37 44.08 44.72 44.40 44.15 43.42 15.58 8.65
To = 68

t = 10 56.02 54.58 56.36 55.76 56.34 55.86 56.09 11.91 6.62

t = 20 49.35 47.72 49.93 49.66 49.86 49.50 49.60 18.40 10.22

Appendix V. The estimation of AV from ATm.

The specific gravity of mature 'Jonathan' apple fruit was reported
to be 0.807 (25). The soluble solid content of mature 'Jonathan' apple
fruit is 12 to 17%. The specific gravity of 12 and 17% sucrose solu—i
tion are 1.0465 and 1.0678, respectively (13), averaging 1.15715.

The volume of 100 g fruit is estimated as:

100 g/0.807 g cm’3 = 123.916 663
The volume of the flesh of a 100 g fruit is estimated as:
100g/1.05715 g cmf3 = 94.594 cm3
The difference between these two values should provide a reasonably
good estimation of the volume (V1) of the intercellular spaces plus
seed cavity, which is:

123.916 cm3 - 94.594 cm3 = 29.322 cm3
The volume of the seed cavity is also considered, because it is physi-
cally connected to the CIS (14).

The AV's, the maximum available volume for solution infiltration,

are calculated according th AV = ATVl/T1 (Appendix II) and tabulated as

the following:

T1 = 283 T1 = 288 T1 = 293
ATm (°C) AV/(ul) ATm(°C) AV(u1) ATm(°C) AV(ul)
2.50 259 4.75 486 6.62 622
3.86 400 7.21 734 10.22 1022
4.69 486 8.65 880 12.36 1236

67

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10.

11.

12.

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