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Relationship between browning and the activities of
polyphenol oxidase and phenylalanine ammonia lyase in
banana peel during low temperature storage
Thi Bich Thuy Nguyen
a,1
, Saichol Ketsa
a,
*, Wouter G. van Doorn
b
a
Faculty of Agriculture, Department of Horticulture, Kasetsart University, Bangkok 10900, Thailand
b
Agrotechnological Research Institute (ATO), Wageningen University and Research Centre, P.O. Box 17, 6700 AA Wageningen,
The Netherlands
Received 17 December 2002; accepted 8 May 2003
Abstract
Kluai Khai (Musa AA Group) and Kluai Hom Thong (Musa AAA Group) bananas were stored at 6 and 10 8C.
Visible chilling injury (CI) in the peel, mainly browning, occurred at both temperatures, but more so at 6 8C, and
without significant differences between the cultivars. At the time of harvest, total free phenolics in the peel were three
times lower in Kluai Khai than in Kluai Hom Thong fruit, the polyphenol oxidase (PPO) activity in Kluai Khai being
considerably higher and phenylalanine ammonia lyase (PAL) activity much lower. As CI developed, PAL and PPO
activities in the peel increased, and total free phenolics decreased. The decrease in total free phenolic compounds and
the increase in PAL and PPO activities occurred more rapidly at 6 8C than at 10 8C, in both banana cultivars.
Correlations between visible CI and the level of total free phenolics, and between CI and the activities of PPO and PAL,
were all highly significant. The results indicate that low temperature stress induced concerted activities of PAL and
PPO, which resulted in browning. Since the concentrations of free phenolic compounds and the rate of PAL and PPO
activities varied considerably between the two cultivars, but browning did not, the changes in the biochemical
parameters rather than their absolute levels were correlated with peel browning.
#2003 Elsevier B.V. All rights reserved.
Keywords: Banana; Musa AA group; Musa AAA group; Peel discoloration; Total free phenolics; Phenylalanine ammonia lyase;
Polyphenol oxidase; Low temperature storage
1. Introduction
Several tropical and subtropical fruit develop
chilling injury (CI) when exposed to low tempera-
tures. Banana fruit is extremely sensitive to CI, i.e.
they show some symptoms at relatively high
temperatures (12 8C) and at lower temperatures
the symptoms are serious. Because of their high
* Corresponding author. Tel.: /66-2-579-0308; fax: /66-2-
579-1951.
E-mail address: agrsck@ku.ac.th (S. Ketsa).
1
This paper is to be referred to as Nguyen et al.
Postharvest Biology and Technology 30 (2003) 187 /193
www.elsevier.com/locate/postharvbio
0925-5214/03/$ - see front matter #2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0925-5214(03)00103-0
sensitivity to chilling, the optimum storage tem-
peratures for banana are 14 /16 8C. The CI
symptoms in banana are usually apparent only in
the peel, where they develop rapidly upon expo-
sure to ambient temperature. An early or mild
symptom is darkening of the peel vascular tissues.
More severe symptoms include browning through-
out the peel surface and failure of ripening
(Pantastico et al., 1967).
Oxidation of phenolic substrates by polyphenol
oxidase (PPO) is believed to be a major cause of
the brown discoloration of many fruits and
vegetables (Mayer and Harel, 1979; Va´mos-Vig-
ya´zo´, 1981; Vaughn et al., 1988; Walker and
Ferrar, 1998). The degree of browning in banana,
after cutting, was correlated with PPO activity and
the concentration of free phenolic substrates
(Weaver and Charley, 1974; Jayaraman et al.,
1982). According to Yang et al. (2001) the main
PPO in banana peel is catechol oxidase (EC
1.10.3.1, o-diphenol: oxygen oxidoreductase).
The enzyme showed two bands after staining
with Coomassie brilliant blue on a PAGE gel,
one very clear and the other faint.
Thus far, no data have apparently been pub-
lished on phenylalanine ammonia lyase (PAL)
activity in banana peel browning, although PAL
is a key enzyme in phenolic metabolism (Va´mos-
Vigya´zo´, 1981). PAL is the first enzyme in the
phenylpropanoid pathway, which produces trans -
cinnamate and trans -4-hydroxycinnamate. These
can be converted (via coumarate: CoA ligase and
hydroxycinnamoyl CoA: quinate hydroxycinna-
moyl transferase) to other phenolic compounds
such as chlorogenic acid and caffeic acid deriva-
tives. In several tissues, the latter are the predo-
minant free phenolics, and they are thought to
serve as substrates for the browning reactions
(Lattanzio et al., 1989; Tomas-Barberan et al.,
1997). In cut lettuce, where browning is related to
a wounding-induced increase in PAL activity,
treatment with inhibitors of PAL activity reduced
browning, and prevented accumulation of free
phenolic compounds. This indicated a causal
relationship between PAL activity and browning
(Peiser et al., 1998; Hisaminato et al., 2001).
We studied the effect of low temperature on
PPO and PAL activities in two commercial banana
cultivars. Chitrakoolsup (1982) showed that these
two cultivars (‘Kluai Khai’ and ‘Kluai Hom
Thong’) were more sensitive to CI than several
other banana cultivars grown in Thailand.
2. Materials and methods
2.1. Plant material
Kluai Khai (Musa cavendishii [Musa acuminata ]
AA Group) and Kluai Hom Thong (Musa AAA
Group) bananas were harvested at commercial
maturity, in a plantation in the Petchaburi pro-
vince (Western Thailand). Bunches were placed in
corrugated cardboard boxes and transported by
refrigerated truck (25 8C) to the laboratory within
2 h of harvest. In the laboratory they were
dehanded and hands were selected for uniformity
of size and colour, then cleaned in a solution of
0.5% MgSO
4
to remove latex from the cut surface.
The fruit were then dipped for 2 /3 min in 500 mg
ml
1
thiabendazole solution to control fruit rot
and allowed to dry at ambient temperature.
Bananas were randomly placed in corrugated
cardboard boxes and stored at 6 and 10 8C, 85%
RH. Fruit were randomly sampled every 3 days for
determination of visible CI symptom, total free
phenolics and activities of PPO and PAL.
2.2. CI assessment
CI in banana fruit was scored visually. The
change in colour in ‘Kluai Khai’ and ‘Kluai Hom
Thong’ bananas, upon chilling, is different from
the dark browning observed during CI in other
fruit such as lychee. It is a rather grey brown.
A rating scale from 1 to 5, based on the intensity
of surface browning, was used: score 1/no CI;
score 2/mild injury; score 3/moderate injury;
score 4/severe injury; score 5/very severe
injury. At score 2 there is no visible browning on
the skin surface but if the epidermal tissues are
peeled, grayish areas are found close to the
surface. Cross-section of the peel shows that these
areas are globular in shape. At score 3 there are
more such areas, which are larger and darker, and
several form continuous lines. At score 4 grayish
T.B.T. Nguyen et al. / Postharvest Biology and Technology 30 (2003) 187 /193188
brown patches are visible on the skin. Cross-
section of the peel shows larger and darker areas
than at score 3. At score 5 there are relatively large
dark patches on the skin surface.
2.3. Total free phenolics and activities of PAL and
PPO
Total free phenolic content was estimated color-
imetrically, using the method described by Single-
ton and Rossi (1965). Briefly, frozen tissue was
homogenized in ethanol, filtered and centrifuged.
The compounds reacted with the Folin Ciocalteau
reagent, and were determined photometrically.
PAL was extracted and assayed by the method
described by Zucker (1968). Frozen tissue was
homogenized in acetone, filtered, extracted again
in cold ethanol, and filtered again. Acetone
powder was dried in a desiccator, and added to
cold 0.2 M sodium borate buffer at pH 8.8. The
beaker was shaken for 30 min in a cold room. The
suspension was filtered and centrifuged. During
the preparation of the enzyme extract, the tem-
perature was kept at 4 8C. The assay medium
contained 3 ml of enzyme extract and 2 ml of
phenylalanine. The mixture was incubated at 37 8C
for 1 h. The reaction was stopped by adding 0.5 ml
of 5 N HCl. PAL activity was determined by
measuring absorbance at 290 nm. One unit of PAL
activity was defined as the change in absorbance
per milliliter enzyme extract.
PPO was extracted and assayed using the
method of by Luh and Phithakpol (1972). The
extraction method was the same as that of PAL,
except that 0.1 M citric buffer at pH 6.2 was used
instead of the borate buffer. The assay medium
contained 10 ml of enzyme extract and 5 ml of
catechol. PPO activity was determined by measur-
ing absorbance at 420 nm. One unit of PPO
activity was defined as the change in absorbance
after 1 min of measurement, per milliliter enzyme
extract.
Protein content in the enzyme extracts was
estimated using the Bradford (1976) method.
Specific activity of the enzyme was expressed as
units per milligram protein.
2.4. Statistical analysis
Four hands were used for assessment of CI. For
measurement of total free phenolics, PAL and
PPO, we took peel from the middle part of ten
fingers from one hand. The peel was taken from
the whole circumference of the fruit, and was 4 cm
long. This was replicated with two other hands.
The peel of the ten fingers from each hand was
mixed.
Free phenolics were determined in fresh tissue,
in the three biological replications, and absorbance
was determined three times from each extract. For
PAL and PPO analysis the acetone powder of peel
from each of the three hands was extracted.
Absorbance was measured four to five times.
Where possible, means were compared using
Duncan’s new multiple range test and Wilcoxon
rank-sum test. Correlations were calculated by
using Pearson correlation coefficients.
3. Results
3.1. Chilling injury
CI was severe in both banana cultivars stored at
68C(Fig. 1). The peel showed discoloration
already on day 3. Between day 3 and 6, there
was a significant increase in browning in both
cultivars. The peel was dark brown after 9 days
storage at 6 8C and fruit showed abnormal ripen-
ing and decay upon transfer to ambient tempera-
ture (data not shown). Therefore, no more
observations were made after 9 days. When stored
at 10 8C, the CI symptoms in the peel did not show
until day 6 (Fig. 1). In both cultivars, the
symptoms were mild until day 12 and slowly
increased thereafter.
The Wilcoxon Rank-sum test showed no
significant difference (P/0.05) in visible
CI between Kluai Khai and Kluai
Hom Thong bananas, at both storage
temperatures.
T.B.T. Nguyen et al. / Postharvest Biology and Technology 30 (2003) 187 /193 189
3.2. Total free phenolics and activities of PPO and
PAL
At the time of harvest, total free phenolics
content of banana peel cv. Kluai Khai was much
lower than that of cv. Kluai Hom Thong. The
total free phenolics level of both banana cultivars
decreased during storage and this decline was
similar (in absolute terms) in both cultivars.
When stored at 6 8C, the total free phenolics level
of both cultivars decreased more rapidly than that
in fruit stored at 10 8C(Fig. 2).
At the time of harvest, PPO activity in banana
peel of cv. Kluai Khai was almost twofold higher
than that of cv. Kluai Hom Thong. PPO activity in
both cultivars increased during storage. This
increase was more rapid at 6 8C than that at
10 8C, in both cultivars. After 9 days of storage,
the PPO activity in cvs. Kluai Khai and Kluai
Hom Thong stored at 6 8C was about 1.1 and 0.7
mg/g protein, respectively, while PPO activity in
banana peel stored at 10 8C reached a similar level
by day 15 (Fig. 3).
At harvest, PAL activity was higher in Kluai
Hom Thong than in ‘Kluai Khai’ bananas, and the
difference remained during storage. PAL activity
in the peel of both cultivars increased during
storage, the increase being greater in fruit stored
at 6 8C than in fruit held at 10 8C. During the first
9 days of storage, the PAL activity in the peel of
cv. Kluai Hom Thong bananas increased more
rapidly than that in cv. Kluai Khai, although the
difference was small in fruit stored at 10 8C(Fig.
4).
A highly significant (PB/0.01) correlation ex-
isted between CI development and total free
Fig. 1. Development of CI of banana peel in cvs. Kluai Khai
(I) and Kluai Hom Thong (b) fruit stored at 6 and 10 8C.
Vertical bars indicate S.D. No bar is shown when S.D. equals
zero.
Fig. 2. Total free phenolics of banana peel in cvs. Kluai Khai
(k) and Kluai Hom Thong (I) fruit stored at 6 and 10 8C.
Vertical bars indicate S.D. No bar is shown when S.D. equals
zero.
T.B.T. Nguyen et al. / Postharvest Biology and Technology 30 (2003) 187 /193190
phenolics, and between CI and the activities of
PPO and PAL (Table 1).
4. Discussion
We found no significant difference in CI sus-
ceptibility between the cultivars Kluai Khai and
Kluai Hom Thong (Fig. 1), although there was a
significant difference in total free phenolic content
between the two cultivars. In both cultivars the
concentration of total free phenolic compounds
decreased during storage, more so at 6 than at
10 8C(Fig. 2). This decrease was therefore corre-
lated with the degree of browning. The free
phenolic compounds may have been used as
substrates for the browning reaction.
PPO activity may be a main factor in the
browning reaction. PPO enzymes have often been
found to localize to the chloroplasts, where they
are associated with the internal thylakoid mem-
branes. They are also found in the cytoplasm and
in vesicles between the plasmalemma and cell wall
(Obukowicz and Kennedy, 1981). Free phenolics
are present mainly in the vacuole, but are synthe-
sized in the cytoplasm (Va´mos-Vigya´zo´, 1981;
Walker and Ferrar, 1998), and perhaps may also
become deposited in the cell walls. If CI induces
membrane damage of organelles such as vacuoles,
the vacuolar phenolics may become in contact
with PPO. Alternatively, the cells may react to
chilling by depositing phenolic compounds in the
cell walls, which would then react with PPO
already present in the apoplast.
Gooding et al. (2001) isolated a number of PPO
(catechol oxidase) cDNA clones from banana
fruit. Levels of the PPO transcripts were low
throughout ripening, in both flesh and peel. The
result suggested that the browning of banana fruits
during ripening resulted from the release of the
Fig. 3. PPO activity of banana peel in cvs. Kluai Khai (k) and
Kluai Hom Thong (I) fruit stored at 6 and 10 8C. Vertical bars
indicate S.D. No bar is shown when S.D. equals zero.
Fig. 4. PAL activity of banana peel in cvs. Kluai Khai (k) and
Kluai Hom Thong (I) fruit stored at 6 and 10 8C. Vertical bars
indicate S.D. No bar is shown when S.D. equals zero.
T.B.T. Nguyen et al. / Postharvest Biology and Technology 30 (2003) 187 /193 191
pre-existing PPO enzyme, which is synthesized
very early during fruit development. Whether this
is also true for banana peel browning at low
temperatures is not clear.
PAL converts phenylalanine to mono- and di-
phenols, which are substrates of PPO. (Tomas-
Barberan et al., 1997). Increased PAL activity has
been related to stress-induced disorders, including
CI (Parkin et al., 1989). In our study, PAL activity
markedly increased at low temperature storage,
particularly at 6 8C(Fig. 4). A high correlation
coefficient was found between changes in PAL
activity and CI development, which may indicate a
causal relation. The activities of PAL and PPO
were also strongly correlated, which indicates
concerted action. A strong inverse relationship
was also observed between PAL activity and total
free phenolics. The turnover of free phenolic
compounds (by PPO and other enzymes) was
apparently more rapid than free phenolic synth-
esis.
Temperatures that induce CI in banana peel
apparently activate PAL. This has also been
described in other cases of CI, for example in
mandarin peel (Martinez-Tellez and Lafuente,
1997). It is not clear whether the increase in PAL
activity in banana peel occurs at the level of
transcription or translation, or after translation.
In citrus, low temperature (2 8C) induced damage
in the fruit flesh, and PAL activity was highly
activated. Northern blot analyses showed that
PAL mRNA accumulated prior to the appearance
of physical chilling symptoms and the accompany-
ing increase in PAL activity (Sanchez-Ballesta et
al., 2000). In this tissue, therefore, the increase in
PAL activity was at least partially due to increased
transcription.
Although browning and an increase in PAL
activity after cutting lettuce was found, suggesting
that PAL was involved in browning (Hisaminato
et al., 2001), the increase in PAL during chilling
may also relate to a defence mechanism. At least in
citrus fruit, the increase in PAL activity after
placement at low temperatures seemed a mechan-
ism to reduce the CI symptoms (Lafuente et al.,
2001). This means that the precise role of PAL
during storage of banana at chilling temperatures
has as yet not fully been established.
In the two banana cultivars studied, the change
in peel PPO activity was also related to the degree
of peel browning, which may suggest that CI-
induced browning is causally related to PPO
activity. Although PPO activity has been measured
during banana browning at higher temperatures
(Jayaraman et al., 1982), this is apparently the first
report showing an increase in banana PPO activity
at chilling temperatures.
It is concluded that the two banana cultivars
studied were sensitive to CI. When the two
cultivars are considered separately, in each of
them CI development in the peel was highly
inversely correlated with the level of free phenolic
compounds, and highly positively correlated with
the activities of PPO and PAL. PAL activity in
banana peel browning is apparently activated in
concert with the activation of PPO. The concen-
trations of free phenolic compounds and the basal
rate of PAL and PPO activities varied consider-
ably between the two cultivars. As the browning
was the same, the change in PAL and PPO activity
Table 1
Relationship between CI development and total free phenolics, and with the activities of PPO and PAL in the peel of the banana cvs.
Kluai Khai and Kluai Hom Thong, stored at 6 and 10 8C
Cultivar Total free phenolics PPO activity PAL activity
K. Khai 6 8C/0.821* 0.958* 0.848*
K. Khai 10 8C/0.711* 0.802* 0.840*
K. Hom Thong 6 8C/0.842* 0.944* 0.960*
K. Hom Thong 10 8C/0.729* 0.650* 0.782*
Data are correlation coefficients.
* Significance at PB
/0.01.
T.B.T. Nguyen et al. / Postharvest Biology and Technology 30 (2003) 187 /193192
rather than their base level was correlated with
peel browning.
Acknowledgements
The authors are thankful for the financial
support from the Ford Foundation and the Thai-
land Research Fund.
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