THE
AGRICULTURAL
SCIENTIST
TissuePHILIPPINE
Hardening in Papaya
(Carica papaya
L.)
Vol. 88 No. 2, 157-166
June 2005
M. Bacay-RoldanISSN
and E.0031-7454
P. Serrano
Etiology and Bases of Tissue Hardening in Heat-Treated
Papaya (Carica papaya L.) Fruits
Marissa Bacay-Roldan1 and Edralina P. Serrano2*
Supported by a grant from the Southeast Asian Regional Center for Graduate Study and Research in Agriculture (SEARCA).
Portion of the doctoral dissertation of the senior author.
1Mindoro
State College of Arts and Trades, Victoria, Mindoro Oriental, Philippines
2Postharvest Horticulture Training and Research Center, College of Agriculture, University of the Philippines Los Baños,
College, Laguna 4031, Philippines
*Author for correspondence; e-mail: [email protected]
The development and mechanism involved in the formation of tissue hardening in heat-treated fruits
of ‘Solo’ papaya (Carica papaya L.) were investigated. Fruits were subjected to hot water treatment
(49 C for 120 min) to induce the development of hardened tissues.
Tissue hardening was more prominent in the inner tissues (-1.5- to 2.0-cm diameter) surrounding
the seed cavity. It was accompanied by a decrease in total sugars and ion leakage. 1aminocyclopropane-1-carboxylic acid (ACC) oxidase activity was inhibited in tissue-hardened fruits,
resulting in the accumulation of ACC but with a reduction in ethylene production. The suppressed
ethylene production inhibited or reduced other ripening-related processes, ultimately causing a 2-d
delay in color change of the peel, tissue hardening or reduced firmness and a concomitant decrease
in pulp color development. On the other hand, the respiration process was not significantly altered,
indicating that O2 and CO2 levels were not involved in tissue hardening.
Polygalacturonase (PG) and xylanase decreased since these are heat-sensitive, thus, dissolution of the middle lamella and destruction of the cell wall were inhibited, and membrane integrity of
hardened tissues was maintained as shown by a reduced ion leakage and viscosity as the fruits
ripened. Xylanase was involved in the softening of papaya fruit up to peel color index 3 stage, while
PG continued to play an important role in the degradation of cell wall components after xylanase
activity declined.
Key words: Carica papaya L., ethylene, hotwater treatment, ion leakage, papaya, polygalacturonase, respiration, tissue
hardening, xylanase
Abbreviations: ACC – 1-aminocyclopropane-1-carboxylic acid, CWDE – cell-wall degrading enzyme, C2H4 – ethylene,
EDTA – ethylene diamine tetraacetic acid, EFE – ethylene forming enzyme, GC – gas chromatography, HWT – hot water
treatment, PCI – peel color index, PG – polygalacturonase, PHTRC – Postharvest Horticulture Training and Research
Center, TA – titratable acidity, TRS – table ripe stage, TSS total soluble solids, WSP – water soluble polyuronide, UPLB
– University of the Philippines Los Baños
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
157
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
INTRODUCTION
Papaya (Carica papaya L.) is one of the major tropical
fruits exported by the Philippines to Japan, Korea and the
Middle East. Strict quarantine regulations must be followed
for exports especially to Japan and Korea. Thus, papaya
fruits must be subjected to appropriate treatment to control fruit fly. In the 1980s, fruits were fumigated with ethylene dibromide, but this chemical has been suspected to
be a carcinogen and an ozone depleter and was ultimately
banned in Japan. Subsequently, exporters tried to disinfest
the fruits by heat treatment.
Heat treatment could be done by exposure to hot water, saturated hot air or vapor heat (Jones 1940; Suzuki et
al. 1994), hot dry air (Tsang and Fuji 1992), infrared radiation, and microwave radiation. All have been used experimentally for fruits and vegetables but vapor heat and
hot water treatment were considered the most practical
(Couey 1989). Vapor heat treatment requires 16- to 18-h
time and greatly reduces decay problems but has frequently
reduced fruit quality (Arisumi 1956).
Tissue hardening in papaya following heat treatment
was attributed to the inhibition of cell-wall-degrading enzymes such as polygalacturonase (PG) and pectin
methylesterase (PME) (Chen and Paull 1986). Although
tissue hardening in other fruits has been associated with
inhibition of cell-wall-degrading enzymes by heat treatment, it was not observed in ‘Solo’ papaya fruits (Ortiz
1994), suggestive of the role of other enzymes in the occurrence of this phenomenon. Our study looked into the
development and bases of tissue hardening including the
involvement of xylanase and polygalacturonase, ethylene
production and respiration in the development of hardened
tissues in papaya fruits as well as changes in their cell walls
after prolonged heat treatment.
MATERIALS AND METHODS
Sound ‘Solo’ papaya fruits with a tinge of yellow color at
their stylar ends (breaker stage) and weighing about 500 g
each were procured from Oriental Mindoro, the Philippines.
Each fruit was wrapped with newspaper, placed carefully
in kraft boxes and immediately transported to the
Postharvest Horticulture Training and Research Center,
University of the Philippines Los Baños (PHTRC, UPLB).
Fruits were then divided into 2 groups, one for control and
one for hot-water treatment. For the hot-water treatment,
the fruits were placed in a net bag and then dipped in thermostat-controlled water bath at 49 C for 120 min. A total
of 60 fruits were used in each of the three experiments,
using a completely randomized design with 3 replicates,
with 20 fruits per replicate.
158
Visual evaluation of color was done using a color index (Pantastico et al. 1984) while quantitative evaluation
was done using a Minolta chromameter. At each measurement, four readings were made at different portions of the
peel. Measurement of pulp color was taken at the center of
the mesocarp.
Firmness of the fruits was measured in terms of the
maximum force (in kg) required for the peel (using the
whole fruit) and pulp (using the cut fruit) to yield to the tip
of the penetrometer attached to an Effegi fruit pressure
tester. Plugs of mesocarp taken near the central portion of
the fruit were sliced thinly into discs for the ion leakage
measurement with a conductivity meter.
Starch was extracted from the alcohol insoluble solids (AIS) and analyzed by the phenol-sulfuric method described by Dubois et al. (1956).
Total sugars was analyzed from the ethanol extract of
the AIS. Aliquots from the extracts were evaporated to dryness with a stream of air, dissolved in a small portion of
water and analyzed using the phenol-sulfuric method
(Dubois et al. 1956).
The static system in conjunction with gas chromatography using the Shimadzu gas chromatograph (GC)
equipped with thermal conductivity detector (TCD) and
silica gel column was used in determining respiration rates
(mg CO2 evolved/kg/h) at each color index. A fruit was
enclosed in a respiration jar for 1 h, after which a 1-mL
headspace gas was withdrawn. This was then injected into
the GC for the measurement of carbon dioxide.
The same setup was used in determining ethylene
(C2H4) production using a Shimadzu GC equipped with a
flame ionization detector (FID) and an activated alumina
column. GC conditions were: column temperature = 110;
injection port temperature = 180 C.
ACC (1-aminocyclopropane-1-carboxylic acid) extraction and assay procedure followed a modified method
described by Lizada and Yang (1979).
The extraction and assay of ACC oxidase followed a
modified procedure described by Ververidis and John
(1991). The standard assay medium consisted of 70 mM
Tris-HCL buffer at pH 7.2, 7% glycerol, 1 mM ACC, 30
mM sodium ascorbate and 100 µM FeSO4 in a total volume of 2 mL including the enzyme. The mixture was incubated in a tightly-capped vacuotainer tube for 30 min at 30
C after which a 1-mL gas sample was withdrawn from the
headspace and analyzed for ethylene by GC.
A modified method described by Saltveit (1982) was
adopted for internal gas analysis. A long, hollow hypodermic needle was inserted through the blossom end into the
cavity of the fruit to extract the gas sample. The concentrations of carbon dioxide, oxygen and ethylene present
within the seed cavity were determined using GC.
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
Tissue Hardening in Papaya (Carica papaya L.)
Extraction of polygalacturonase (PG) and xylanase followed a procedure modified from Paull and Chen (1983).
Pericarp tissue was immediately dipped in liquid nitrogen
after weighing. While being thawed, the tissue was homogenized with 2 volumes of 7.5% (w/v) NaCl + EDTA (10:1)
pH 7.0 at 4 C. After standing at 4 C for 15 min, the mixture
was centrifuged at 10,000 g for 10 min. Partial purification of the crude extract from sugars and other impurities
was done by gel filtration using Sephadex G-25. Aliquots
were used for PG and xylanase assays.
PG activity was measured viscometrically following
the procedure modified from Paull and Chen (1983). The
reaction mixture consisted of 2 mL of enzyme solution in
citrate NaCl buffer at pH 4.6 and 10 mL of 1.2% pectin in
0.2 M citrate buffer at pH 4.6.
The pectin-buffer mixture was added to the viscometer and allowed to equilibrate to the bath temperature (45
C). The enzyme was added rapidly to the pectin-buffer
mixture and mixed by bubbling it through the mixture.
Difference in viscosity was determined at zero time and at
20-min intervals. One unit of PG activity was defined as
1% loss in viscosity per hour.
Reducing groups released after 3 h of incubation of
the reaction mixture was also measured as modified from
Honda et al. (1982). One mL of the solution was taken
from the reaction mixture described in the preceding paragraph. The reaction was stopped with the addition of 5 mL
100 mM borate buffer, pH 9.0. The mixture was boiled for
10 min following the addition of 1 mL of 1% 2cyanoacetamide solution. The amount of reducing groups
released was spectrophotometrically measured at 276 nm
and calibrated against a standard curve using galacturonic
acid.
The assay for xylanase activity followed a procedure
modified from Paull and Chen (1983). Xylanase assay solution contained 0.09% (w/v) xylan (Sigma Chemical Co.)
and 0.09% (w/v) casein in 40 mM sodium acetate buffer
(pH 5.0). The reaction mixture contained 0.2 mL enzyme
suspension and a total of 2 mL assay solution. The mixture
was incubated for 1 h at 37 C then terminated with 0.4 mL
2M HCL. After centrifugation at 10,000 x g for 10 min,
the concentration of reducing sugars in the supernatant was
determined spectrophotometrically at 600 nm. Protein content was analyzed by the method of Lowry et al. (1951).
For the anatomical study, 1-cm pieces from the seed
cavity of affected and non-affected tissues were soaked in
3% glutaraldehyde for 1 h and then washed for 15 min.
with buffer five times. The samples were then fixed in 1%
(w/v) osmium tetroxide (OsO4) for 2 h and then washed
with buffer five times. Dehydration was carried out with
ethanol series (50%, 70%, 80%, 90%, 95% and 100%) at
room temperature. Substitution with 50% propylene oxide
(PO) was done for 30 min and then at 100% PO for 1 h.
M. Bacay-Roldan and E. P. Serrano
Peel Color Index
6
a
5
a
b
a
4
a
b
b
3
b
2
1
0
2
4
6
Storage (d)
8
Fig. 1. Peel color index of papaya fruits: control ( O),
subjected to hot-water treatment ( Q). Each data
point represents the average of 3 replicates.
Means with different letters at each day of storage are significantly different at 5% level DMRT.
PCI 1 = green, PCI 2 = tinge of yellow, PCI 3
= more green than yellow, PCI 4 = more yellow than green, PCI 5 = trace of green, PCI 6
= full yellow.
Tissues were then infiltrated with Spurr Resin and then
with PO resin at different proportions (3:1, 1:1 and 1:3)
and, finally, with pure resin. The materials were positioned
in an embedding mixture in an oven (60 C). Semi-thin sections (1 µm) were cut with a glass knife, mounted on glass
slides and stained with toluidine blue with borax. Mounted
sample tissues were viewed under a scanning microscope.
RESULTS
Physio-Chemical Changes
Color changes in peel and pulp. Hot water treatment delayed peel color change (Fig. 1). Untreated fruits showed
normal color change during ripening and became entirely
yellow-orange (PCI 6) in 7 d. On the other hand, it took 9
d for the hot-water-treated fruits to attain PCI 6. Correspondingly, at PCI 6, the chromameter ‘a’ value of the peel
of control fruits was higher at 3.546 compared to -2.633
for the hot-water-treated fruits (Fig. 2A). The pulp showed
the same color changes as that of the peel but the color
difference between the pulp of the treated and untreated
fruits was more obvious. The pulp of the heat-treated fruits
at PCI 6 had a significantly lower ‘a’ value of -0.229, while
that of the untreated fruits had an ‘a’ value of 5.963 (Fig.
2B). An and Paull (1990) also observed the retardation of
skin yellowing of papaya above 30 C.
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
159
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
a value
4
a value
10
a
A
B
8
0
-4
a
6
b
4
-8
2
-12
-16
0
1
2
3
4
5
Peel Color Index
-2
6
b
1
2
3
4
5
Pulp Color Index
6
Fig. 2. (A) Peel and (B) pulp color changes in control ( O) and hot water-treated ( Q) papaya fruits. Each data point represents the average of 3 replicates. Means with
different letters at each peel/pulp color index are significantly different at 5% level
by DMRT. For peel color index equivalents see Fig. 1.
The hardened pulp was prominently lighter in color than
the unaffected pulp of the same fruits (Fig. 3). This was
also observed by Akamine and Seo (1978) and Suzuki et
al. (1991) in heated papaya fruits.
Tissue firmness. Softening of fruit tissues is one of the
most pronounced changes associated with ripening. Figure 4 shows the gradual decline in firmness of the untreated
fruits as ripening progressed. Treated fruit tissues were significantly firmer than the control and increased in firmness values at PCI 3 up to PCI 4 instead of declining. The
first indication of tissue hardening as the fruits ripened was
noted at PCI 3 and this disorder gradually progressed as
the fruits ripened. Approximately, 1.5 to 2.0 cm of hardened tissues were observed near the seed cavity. Eventually, the entire mesocarp hardened except for a very thin
layer immediately under the peel. The hardest tissues were
detected at PCI 5 where tissues were even harder than the
pulp of fruits that were just starting to soften (PCI 2). The
average force required to penetrate the hardened tissue,
which is a measure of firmness, was 5.853 kg, whereas at
PCI 2, it was only 4.77 kg. This tissue hardening was observed in 87% of the treated fruits.
Apart from tissue hardening, another disorder noted
in hot-water-treated fruits was the appearance of watersoaked tissue in 13% of the fruits when the fruits were
about 25% yellow (PCI 3). Tissues rapidly became softer
so that at the full-ripe stage (PCI 6), the force required to
resist tissue penetration was only 0.153 kg.
Ion leakage. An important phenomenon occurring in
most ripening fruits, coincident with loss of tissue firmness, is the increase in ion leakage. Figure 5 shows that at
160
A
Contr
ol
Control
Ho
t-w
at
er
-treat
ed
Hot-w
t-wat
ater
er-treat
-treated
B
Fig. 3. Samples of control and hot-water-treated fruits
showing A) difference in peel color after 3 d of
storage, and B) difference in the appearance
of the pulp. Fruits were hot-water-treated at PCI
2. Hot-water-treated fruits have hardened tissues measuring 1.5 to 2.0 cm in diameter surrounding the seed cavity which is pale in color.
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
Ion Leakage (mS/cm)
Firmness (kg)
7
0.40
6
0.35
a
5
a
a
4
a
a
0.30
b
3
b
0.25
2
b
0.20
b
0.15
1
2
3
4
5
Peel Color Index
6
b
b
b
1
0
a
a
7
1
2
3
4
5
6
7
Peel Color Index
Fig. 4. Firmness in control ( O) and hot-water-treated
papaya with hardened ( Q) and softened ( V) tissues. Except in V, each data point represents
the mean obtained from 3 replicates. Means
with different letters at each peel color index
are significantly different at 5% level by DMRT.
For peel color index equivalents see Fig. 1.
Fig. 5. Ion leakage in control ( O) and hot water treated
papaya with hardened ( Q) and softened ( V) tissues. Except in V, each data point represents
the mean obtained from 3 replicates. Means
with different letters at each peel color index
are significantly different at 5% level DMRT. For
peel color index equivalents see Fig. 1.
PCI 2, a relatively low ion leakage, 0.235 mS/cm, was observed in untreated fruits. The value continuously increased
- at PCI 3, by 25.55% and at PCI 6, by almost 100%. With
gradual decrease in firmness of the untreated fruits from
PCI 2 to PCI 6, a corresponding increase in ion leakage
became more noticeable.
The treated fruits with hardened tissues showed a general decrease in ion leakage. At PCI 4, ion leakage decreased by 32.34% compared with that at PCI 2. At PCI 5,
when the mesocarp was observed to be hardest, ion leakage was lowest. Abnormal softening was accompanied by
an increase in ion leakage. The difference in conductivity
value between treatments was significant at 5% level.
Sugars. Percentage total sugars in papaya substantially
increased as the fruits ripened and this was more apparent
in the untreated fruits (Fig. 6). At the start of the study, the
percentage of sugar in the untreated and the treated fruits
were 7.04% and 7.36%, respectively. Subsequently, there
was a gradual increase in the percentage of sugar in both
treatments, but that of the treated fruits remained relatively
lower and had its peak at PCI 4. At the table ripe stage,
total sugar content in the untreated fruits (10.07%) was
19.84% higher compared to the treated fruits.
The contribution of starch breakdown to textural and
flavor changes during ripening may be negligible since papaya fruits have no starch reserves for the production of
soluble sugars after harvest (Chan et al. 1979). Throughout the storage period, there was no significant difference
in the starch content of both untreated and treated papaya
(data not shown). Starch content ranged from 0.214% to
0.631% and no trend was observed during ripening.
Physiological Changes
Respiration. Papaya fruits that were not subjected to hotwater treatment exhibited the typical climacteric pattern of
respiration (Fig. 7). At PCI 2, the average respiration rate
was 69.11 mg CO2 kg-1h-1 and this value slightly decreased
up to PCI 4. Thereafter, the fruit demonstrated a characteristic burst in CO2 production and the respiratory climacteric appeared to peak at a relatively advanced stage of
ripening (PCI 5). During this peak, the respiration rate was
78.12 mg CO2 kg-1h-1 which was within the range observed
in previous studies in papaya (Angeles 1984; Paull and Chen
1983; Aziz et al. 1975).
Respiration rate in hot-water-treated fruits was significantly higher (126.91 mg CO2 kg-1h-1) during the initial
analysis immediately after treatment. As the temperature
of the fruits dropped back to ambient level, respiration at
PCI 3 and PCI 4 also decreased to a level comparable to
that of the untreated fruits. Respiration rate for both control and heat-treated fruits increased at PCI 5, which was
the climacteric peak, but this increase continued until the
full-yellow stage (PCI 6) for heat-treated fruits.
Internal CO2 and O2 levels were not affected by heat
treatment (data not shown). It can be surmised that these
gases are not involved in the development of tissue hardening.
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
161
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
Respiration Rate mg CO2.kg-1.h-1
% Sugar
11
140
a
120
10
100
9
8
7
60
a
40
a
20
6
1
2
3
4
Peel Color Index
5
6
7
a
b
80
1
2
3
4
b
5
6
Peel Color Index
Fig. 6. Percent (%) sugar in control ( O) and hot water
treated ( Q) papaya at different stages of ripening. Each data point represents the mean obtained from 3 replications. No significant differences in % sugar content were observed at
each peel color index at 5% level DMRT. For
peel color index equivalents see Fig. 1.
Fig. 7. Respiration rate in control ( O) and hot watertreated ( Q) papaya fruits at different stages of
ripening. Each data point represents the mean
obtained from 3 replicates. Means with different letters at each peel color index are significantly different at 5% level by DMRT. For peel
color index equivalents see Fig. 1.
ACC oxidase activity, ACC, ethylene production and
internal ethylene. ACC oxidase activity was significantly
inhibited by heat treatment (Fig. 8A). While both untreated
and treated fruits had the peak of enzyme activity at PCI 4,
leveling off at PCI 5 and 6, the enzyme activity was remarkably higher in the untreated fruits by 45.62% compared to the treated fruits. At the full-yellow stage, the difference increased to 70.94%. In general, there was a considerably higher amount of ACC in the treated fruits than
in the untreated fruits except at PCI 5 (Fig. 8B), but the
differences at each PCI were not significant.
Ethylene production was significantly higher in the untreated fruits after PCI 2 (Fig. 8C). The rate has more than
doubled when it peaked at PCI 4, then decreased up to PCI
6. Ethylene production at PCI 6 was much higher than during the preclimacteric stage.
While a burst in ethylene production was very noticeable at PCI 4 in the control fruits, there was a significant
lag in ethylene production in the treated fruits. From an
initial 0.324 µL, this value gradually declined to 0.1362
µL C2H4/kg/h at PCI 6.
In the untreated fruits, internal ethylene within the seed
cavity correspondingly increased (Fig. 8D). At PCI 3, a
very minimal amount of ethylene (0.176 ppm) was evolved.
At PCI 5, it was almost ten times as much. At full yellow,
there was a decrease by 16%. This is in consonance with
the finding of Akamine and Goo (1979). The decline at
full-yellow coincided with the decrease in ACC oxidase
activity.
The internal ethylene in the treated fruits was significantly lower than that in the untreated fruits except at PCI
3. It remained at a very minimal amount up to PCI 6, suggesting that ethylene production was indeed inhibited. At
PCI 5, ethylene level in the seed cavity of the untreated
fruits was seven times higher than that in the treated fruits.
162
Biochemical Changes
Xylanase. Significant differences in the xylanase activity
was noted between the untreated and the treated fruits with
hardened tissues (Fig. 9). In all fruits, a very minimal activity at color break was followed by an upsurge in activity at PCI 3, which subsequently declined.
The peak of xylanase activity at PCI 3 of the treated
fruits was significantly lower than that of the untreated
fruits. Xylanase activity declined at PCI 4. Entirely no activity of the enzyme was detected in both treatments at PCI
6. Xylanase activity in the softened tissues of the treated
fruits was similar to that of the control.
Polygalacturonase activity. Figure 10 shows the polygalacturonase activity, which is the activity of the enzyme that catalyzes the hydrolytic cleavage of alpha (1-4)
galacturonan linkages and, hence, is primarily responsible
for the dissolution of the middle lamella during ripening.
A low activity occurred at the breaker stage. A marked increase was noted at PCI 3 that gradually increased until
the full yellow stage. The increase was significantly higher
in the untreated fruits compared with the treated fruits.
There was a suppressed PG activity in the treated fruits,
suggesting a remarkable inhibition of the enzyme by heat
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
ACC oxidase activity (nmol C2H4/g tissue)
30
a
AA
a
25
Ethylene (mL/kg/h)
1.0
B
B
20
0.6
b
15
a
0.8
a
0.4
10
b
b
b
b
0
0.0
ACC (nmol C2H4/g tissue)
3
CC
Internal C2H4 (ppm)
2.5
DD
a
2.0
a
2
1.5
a
1.0
1
b
0.5
0
b
0.2
5
a
2
3
4
5
Peel Color Index
6
0.0
7
b
2
3
b
4
5
Peel Color Index
6
7
Fig. 8. A) ACC oxidase activity, B) ACC, C) ethylene production and D) internal ethylene in
control ( O) and hot-water-treated ( Q) papaya fruits. Each data point represents
the mean of 3 replicates. Means with different letters at each color index are
significantly different at 5% level by DMRT. For peel color index equivalents see
Fig. 1.
Xylannase activity (mg Glucose/mg protein/h)
60
50
a
40
30
b
a
20
b
10
0
1
2
3
4
Peel Color Index
5
6
Fig. 9. Xylanase activity of control ( O) and hot-watertreated fruits with hardened ( Q) and softened
( V) tissues. Except in ²%, each data point represents the mean obtained from 3 replicates.
Means with different letters at each color index
are significantly different at 5% level by DMRT.
For peel color index equivalents see Fig. 1.
treatment. Lu et al. (1990) suggested that PG played a major
role in ripening fruits and that regulation of fruit ripening
by ethylene is mediated by PG.
Viscosity loss. Viscosity loss was greatest at PCI 4,
quite ahead of the observed climacteric (Fig. 11). At PCI
3, this activity more than doubled compared to PCI 2 in
the untreated fruits. At peak, increase in viscosity loss was
more than three-fold. This was followed by a rapid decline, which corresponds to 63% of the peak value.
Treated fruits at PCI 3 did not attain a 50% viscosity
loss, which was significantly lower than that of the untreated
fruits. The viscosity loss in softened tissues of the treated
fruits did not vary significantly from those of the ripe tissues of the untreated fruits although the level was lower.
Changes in the Cell Wall
At the fully ripe stage (PCI 6), cell walls in the unheated
fruits were no longer clearly defined as there was complete dissolution of the middle lamella and the cell walls.
Only the vascular tissue (vt) remained evident (Fig. 12A).
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
163
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
PG Activity (nmol Galacturonic acid/ml)
% Viscosity Loss
1600
70
a
1200
a
50
a
b
b
b
600
a
20
b
10
1
2
3
4
5
6
7
Peel Color Index
b
On the other hand, in the heat-treated fruits, a wellordered arrangement of parenchyma cells (pc) in the mesocarp tissue of the fruits was still present even at PCI 6
(Fig. 12B). Darkly stained, rigid cell walls remained evident. Cells were observed to be more round in shape compared to those at PCI 2 and PCI 4 and remained intact.
Cell walls of adjoining cells were observed to be closely
linked to each other. Middle lamella could still be observed
in between cell walls. Hence, cell wall integrity was maintained. This characteristic of the cell wall accounted for
the hardening of the tissues of the heat-treated fruits.
DISCUSSION
Hot-water treatment of ‘Solo’ papaya at 49 C for 120 min
resulted in hyperthermal injury observed in the hardening
of its pulp more prominently near the seed cavity, the lighter
color of the peel and pulp and 2 d delay in color change
(Fig. 1, 2). The fruits ripened externally but remained unripe internally. The observed tissue hardening (Fig. 3) with
prolonged heat treatment was also observed by Akamine
and Seo (1978) and Suzuki et al. (1991).
Changes in the physiological, biochemical and anatomical processes in the ripening fruits accompanied tissue hardening, which was first noted at PCI 3. Notable
among these changes were those of the rate of ethylene
production and activities of two cell-wall-degrading enzymes, polygalacturonase and xylanase.
Ethylene production was inhibited at all stages of ripening so that internal ethylene was lower (Fig. 8). The res-
a
b
0
1
Fig.
ig. 10. PG activity of control ( O) and hot-water-treated
fruits with hardened ( Q) and softened ( V) tissues. Except in V, each data point represents
the mean obtained from 3 replicates. Means
with different letters at each peel color index
are significantly different at 5% level DMRT.
For peel color index equivalents see Fig. 1.
164
b
30
b
400
a
40
1000
800
a
60
a
1400
2
3
4
Peel Color Index
5
6
7
Fig. 11. Percent viscosity loss in control ( O) and hotwater-treated fruits with hardened ( Q) and
softened ( V) tissues. Except in V, each data
point represents the mean obtained from 3
replicates. Means with different letters at
each color index are significantly different at
5% level DMRT. For peel color index equivalents see Fig. 1.
piration process was not significantly altered except immediately after treatment (Fig. 7) due to the increased temperature and at the full yellow stage when disease started
to invade the tissues. In fruits which ripen normally, internal oxygen is depleted when the fruits are about 50% yellow (Selamat 1993) and the concentration of CO2 increases
as the fruit ripens (Lazan and Zim 1990; Akamine and Goo
1979). In this study, the heat-treated fruits, internal CO2
and O2 were not affected and were thus not involved in the
development of tissue hardening.
ACC oxidase catalyzes the oxidation of ACC to ethylene (Kasai et al. 1996). When ACC oxidase activity was
at the maximum, ACC level was low (Fig. 8), suggesting
that ACC was used up as substrate by ACC oxidase in synthesizing ethylene. Inhibition of ACC oxidase results in
the accumulation of ACC but reduces ethylene production.
The significantly low activity of ACC oxidase confirms
earlier reports that in heat-treated fruits, the ethylene-forming enzyme (ACC oxidase) system is labile, possibly membrane-bound (Apelbaum et al. 1981) and heat-sensitive (Yu
et al. 1980).
The activity of xylanase, a cell-wall degrading enzyme
involved in softening (Paull and Chen 1983; Labavitch and
Greve 1983), was disrupted by heating. Xylanase activity
was lower in the treated fruits (Fig. 9). Paull and Chen
(1983) also observed that tissue hardening in papaya was
accompanied by a marked reduction in xylanase activity.
In this study, xylanase was active only at PCI 3 and 4, with
peak of activity at PCI 3. The xylanase activity of the softer
portions of the treated fruits was similar to that of the con-
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
Tissue Hardening in Papaya (Carica papaya L.)
trol. A linear relationship between xylanase activity and
loss of tissue firmness could not be established. Apparently, xylanase is involved in fruit softening up to PCI 3
after which other cell-wall-degrading enzymes, particularly
PG, were responsible for the continued softening of the
fruits. This is substantiated by results on viscosity measurements (Fig. 10), indicating increasing PG activity beyond PCI 3 and an endo-hydrolysis mode of PG action as
shown by viscosity loss results (Fig. 11).
The observed tissue hardening coincident with a
marked reduction in xylanase activity in treated fruits substantiates the concept (Paull and Chen 1983) that xylanase
plays a significant role in softening, but the enzyme is inactivated or deactivated at high temperature either by destroying the enzyme’s activity directly or by preventing its
synthesis (Egusa et al. unpublished).
The suppression of ethylene production in heat-treated
fruits is a crucial factor in the inhibition of other ripeningrelated processes. With the significant inhibition of ethylene production, there was a decreased xylanase activity up
to PCI 3 and PG activity. This phenomenon may have hindered dissolution of the middle lamella and solubilization
of cell wall pectins (Fig. 12), thereby, leaving the cell walls
intact and membrane integrity maintained as shown by a
reduced ion leakage (Fig. 5). This situation might have
caused the failure of the treated fruits to soften although
they were ripe externally.
Tissue hardening was more prominent in the inner tissues surrounding the seed cavity most probably because
ACC oxidase within the mesocarp and the endocarp is more
heat-sensitive than ACC oxidase in the exocarp (Chan
1991). Lazan et al. (1989) also observed the effect of heat
treatment in suppressing PG activity, which was relatively
greater in the inner than in the outer mesocarp tissue. It
resulted in greater inhibition of ethylene production in the
inner portion (endocarp) than in the exocarp. Accordingly,
the synthesis or activity of PG or xylanase was also inhibited.
Inasmuch as ethylene production was inhibited by heat
and that ethylene preceded other ripening-related processes,
color development was delayed. The delayed change in
the peel and pulp color of the heat-treated fruits is directly
due to loss of total chlorophyll and the synthesis of total
carotenoids (Aziz et al. 1975).
Apart from tissue hardening and delayed color development in the treated fruits, a 20% reduction in total sugars at the full-ripe stage (Fig. 7) was also noted. There was
no effect of heat treatment on the conversion of starch into
sugar since there is very little or no starch in papaya. Retention of tissue firmness, therefore, cannot be attributed
to the presence of starch.
M. Bacay-Roldan and E. P. Serrano
Fig. 12. Cells of A) control and B) heat-treated fruits
at PCI 6. In control fruits, parenchyma cells
have no distinct structure. Cell walls have undergone complete dissolution (d-cw) only vascular tissue (vt) can be noted while in hot
water-treated fruits, parenchyma cells (pc) are
still intact, and cell walls (cw) are not yet disrupted x 150; bar = 0.2mm.
CONCLUSION
The study supports the hypothesis that in the development
of tissue hardening, heat treatment inhibits ACC oxidase,
thus lowering ethylene production and causing an accumulation of ACC in the inner mesocarp tissue of the papaya fruits. Inhibited ethylene production coincided with
decreased activities of PG and xylanase, which hindered
the dissolution of the middle lamella and the solubilization of cell wall pectins, thereby, leaving the cell walls intact and membrane integrity maintained, as evidenced by
reduced ion leakage and viscosity.
There are still other aspects of tissue hardening in
‘Solo’ papaya that need to be fully elucidated such as the
formation of water-soluble polyuronides and changes in
ACC levels and ACC synthase. The mechanism of delayed
The Philippine Agricultural Scientist Vol. 88 No. 2 (June 2005)
165
Tissue Hardening in Papaya (Carica papaya L.)
M. Bacay-Roldan and E. P. Serrano
peel and pulp color development in hardened tissues still
need to be elucidated.
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