Reduction of enzymatic browning of harvested ‘Daw’ longan exocarp by sodium chlorite

Abstract

Post-harvest exocarp browning is a major problem resulting in reduced shelf-life of longan fruits. The objective of this study was to evaluate the possibility of using sodium chlorite (SC) as an anti-browning agent for controlling enzymatic browning of harvested longan fruits during storage at ambient conditions. Longan fruits cv. Daw were dipped in 0.001%, 0.005%, 0.01%, and 0.05% SC (W/V) for 10 min. The fruits were packed in cardboard boxes and stored at 25 +/- 1 degrees C with a relative humidity of 82 +/- 5% for 72 h. Changes in browning index, colour parameter (L* and b* values), polyphenol oxidase (PPO) activity, peroxidase (POD) activity, and total phenolic content were measured. The results showed that the fruits treated with SC had lower browning index, but higher L* (lightness) and b*(yellowness) values than those of the control group during storage for 48 h. SC at a concentration of 0.01% was the most effective in reducing exocarp browning. The application of SC reduced PPO and POD activities and delayed a decrease in the total phenolic content. The treatment with 0.01% and 0.05% SC had the lowest PPO and POD activities, and maintained the highest total phenolic content. It was concluded that an application of SC is an alternative method for reducing exocarp browning and maintaining quality of harvested longan fruits.

Full text

R
ESEA RCH ARTI CLE
doi: 10.2306/scienceasia1513-1874.2011.37.234
ScienceAsia 37 (2011): 234–239
Reduction of enzymatic browning of harvested ‘Daw’
longan exocarp by sodium chlorite
Bundit Khunpona, Jamnong Uthaibutraa,b, Bualuang Faiyuec, Kobkiat Saengnila,b,∗
aDepartment of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand
bPostharvest Technology Innovation Centre, Commission on Higher Education, Bangkok 10400, Thailand
cDepartment of Biology, Mahidol Wittayanusorn School, Salaya, Phutthamonthon, Nakhon Pathom 73170,
Thailand
∗Corresponding author, e-mail: kobkiat s@hotmail.com
Received 11 Apr 2011
Accepted 2 Sep 2011
ABSTRACT: Post-harvest exocarp browning is a major problem resulting in reduced shelf-life of longan fruits. The
objective of this study was to evaluate the possibility of using sodium chlorite (SC) as an anti-browning agent for controlling
enzymatic browning of harvested longan fruits during storage at ambient conditions. Longan fruits cv. Daw were dipped
in 0.001%, 0.005%, 0.01%, and 0.05% SC (W/V) for 10 min. The fruits were packed in cardboard boxes and stored at
25 ±1 °C with a relative humidity of 82 ±5% for 72 h. Changes in browning index, colour parameter (L* and b* values),
polyphenol oxidase (PPO) activity, peroxidase (POD) activity, and total phenolic content were measured. The results showed
that the fruits treated with SC had lower browning index, but higher L* (lightness) and b*(yellowness) values than those
of the control group during storage for 48 h. SC at a concentration of 0.01% was the most effective in reducing exocarp
browning. The application of SC reduced PPO and POD activities and delayed a decrease in the total phenolic content.
The treatment with 0.01% and 0.05% SC had the lowest PPO and POD activities, and maintained the highest total phenolic
content. It was concluded that an application of SC is an alternative method for reducing exocarp browning and maintaining
quality of harvested longan fruits.
KEYWORDS:Dimocarpus longan, peroxidase (POD), polyphenol oxidase (PPO)
INTRODUCTION
Longan is a commercial subtropical fruit, widely
grown in China, Thailand, India, and Vietnam1–3. Un-
fortunately, the fruit has a very short post-harvest life
and the visual appeal of longan can deteriorate within
3 days under ambient conditions4–6due to pericarp
browning and breakdown, resulting in reduced market
value7–9. Colour is one of the most important visual
characteristics for marketing longan fruits10. Brown-
ing of longan has mainly been attributed to oxidation
of phenolic compounds by polyphenol oxidase (PPO)
and peroxidase (POD), producing brown-coloured by-
products7,11. Reducing enzymatic browning is im-
portant to extend storage life and maintain quality of
longan fruits12.
Several methods have been used to prevent en-
zymatic browning of fruits and vegetables13 . One
of these is the use of anti-browning agents such as
chitosan11, citric acid, ascorbic acid, oxalic acid 14,
and nitric oxide12. Using these agents is constrained
by their high cost and harmfulness. Consequently,
research and development studies to find effective
substitutes are still ongoing15,16.
Sodium chlorite (SC) is an oxidizing and sanitiz-
ing agent which is able to generate chlorine dioxide
(ClO2) in an acidic environment17 . The American
Food and Drug Administration has approved its use on
raw fruits and vegetables in the concentration range of
0.05% to 0.12%18. It has been reported that SC and
ClO2have been used to reduce enzymatic browning
of fruits and vegetables19–24 . For example, Lu et al19
demonstrated that SC at a concentration of 0.09%
(w/v) significantly inhibited PPO activity extracted
from fresh-cut apples (Malus domestica Borkh. cv.
Red Delicious). Similarly, Lu et al20 found that
dipping in 0.05% (w/v) SC for 1 min inhibited enzy-
matic browning of fresh-cut Red Delicious apples. Fu
et al21 reported that an aqueous solution of ClO2at
0.005% (w/v) inhibited the activity of PPO extracted
from Golden Delicious apples by 63%. In the same
way, Guan and Fan22 reported that dipping in SC at
0.05% (w/v) for 5 min reduced enzymatic browning
and microbial population of fresh-cut Granny Smith
apples. Du et al 23 found that dipping fresh-cut lotus
(Nelumbo nucifera Gaertn cv. Bai Hua) roots in 0.01%
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ScienceAsia 37 (2011) 235
(w/v) ClO2solution for 10 min significantly decreased
the activity of PPO and delayed browning. Chen et
al24 reported dipping in 0.01% (w/v) ClO2for 20 min
inhibited enzymatic browning and extended shelf-life
of fresh-cut asparagus lettuce (Lactuca sativa L. var.
angustana Irish).
Although SC has been reported to reduce brown-
ing in many fruits and vegetables, there is no report
on the effect of SC on enzymatic browning in longan.
The objective of this study was to evaluate the possi-
bility of using SC as an anti-browning agent for longan
fruits during storage at ambient conditions.
MATERIALS AND METHODS
Plant materials
Longan (Dimocarpus longan Lour. cv. Daw) fruits at
commercial maturity were harvested from a commer-
cial orchard in Lamphun province, Thailand, in July
and August 2010 and February and March 2011 for
repetition. Fruits were delivered to a laboratory room
in the Department of Biology, Chiang Mai University,
within 1.5 h. The fruits were individually selected
from a bunch for uniformity of shape, colour, size, and
lack of defects. The fruits were randomly distributed
into five groups of 120 fruits. The fruits were then
dipped in sodium chlorite (SC) solutions at concentra-
tions of 0.001, 0.005, 0.01, and 0.05% (W/V) (pH 5.5)
for 10 min at room temperature (25 ±1 °C). Fruits
dipped in distilled water (pH 5.5) were used as a
control. After dipping, the fruits were air-dried for
10 min, packed in cardboard boxes, and then stored
for 72 h at room temperature with a relative humidity
of 82 ±5%. Fruits from each treatment and control
were randomly sampled at 12, 24, 48, and 72 h after
storage to measure browning index, colour of exocarp,
PPO and POD activities, and total phenolic content.
Browning index
Exocarp browning was estimated visually by measur-
ing the extent of the total brown area on each fruit
surface using the following scale11 : 1=no browning
(excellent quality), 2=slight browning, 3=less than
25% browning of the total surface, 4=25–50% brown-
ing, and 5=>50% browning (poor quality). A
browning index was calculated using the following
formula: P(browning scale ×percentage of fruit
in each class)11. Fruits having a browning index
above 3.0 were considered as unacceptable for visual
marketing quality11.
Colour measurement
The colour of the exocarp was measured using a
chromameter (Model Miniscan XE plus, Germany)
and the degree of browning was expressed as L* and
b* values (CIE 1976). L* values indicated lightness
of the exocarp, ranging from black=0 to white=10010,
whereas b* values indicated classification of yellow to
blue ranging from yellow (>0) to blue (<0)23 .
Extraction of enzymes and assay for PPO and
POD
Enzymes were extracted by the modified method of
Huang et al25. Longan exocarp (2 g) from 20 fruits
was homogenized in 20 ml of 0.05 M potassium
phosphate buffer (pH 6.2) containing 1 M KCl and 2%
polyvinylpyroritidone for 5 min by using a mortar and
pestle, and centrifuged for 5 min at 20 000g(Hermel
model Z383K, Germany) and 4 °C. The supernatant
was then collected for PPO and POD activity assays
as a crude enzyme extract.
PPO activity, using catechol as a substrate, was
assayed based on the method of Jiang and Fu26 using
the reaction mixture (2 ml) containing 1.3 ml of
0.05 M potassium phosphate buffer (pH 7.5), 0.2 ml
of 0.2 M catechol, and 0.5 ml of crude enzyme. Tubes
were incubated for 5 min at 30 °C. The absorbance
was measured at 420 nm in a visible spectropho-
tometer (Model Thermo Spectronic, USA). The unit
of enzyme activity was defined as the amount of
enzyme that caused a change of 0.01 in absorbance
per minute14.
POD activity, using guaiacol as a substrate, was
assayed based on the method of Nagle and Harrd27
using a reaction mixture (2.5 ml) containing 2.3 ml
of 0.01 M sodium acetate buffer (pH 6.0), 0.05 ml
of 0.1% guaiacol (V/V), 0.1 ml of 0.1% H2O2(V/V),
and 0.05 ml of crude enzyme. Tubes were incubated
for 5 min at 30 °C and the absorbance was measured
at 470 nm in a visible spectrophotometer (Model
Thermo Spectronic, USA). The unit of enzyme
activity was defined as explained for PPO activity.
Protein levels were assayed from crude enzyme
extracts according to Lowry et al28 with Folin-
Ciocalteu reagent as a standard.
Determination of total phenolic content
The total phenolic content was determined by the
method of Singleton and Rossi29. Longan exocarp
(2 g) from 20 fruits was homogenized in 20 ml of
80% ethanol for 5 min by using a mortar and pestle,
and then centrifuged for 20 min at 16 000g(Hermel
model Z383K, Germany) and 4 °C. Two hundred
microlitres of clear supernatant were mixed with 10 ml
of 10% Folin-Ciocalteu reagent (V/V) for 8 min.
Then, 8.0 ml of 7.5% sodium carbonate (W/V) was
added. Tubes were incubated for 2 h at 30 °C and
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236 ScienceAsia 37 (2011)
the absorbance was measured at 765 nm in a visible
spectrophotometer (model Thermo Spectronic, USA).
A standard curve of gallic acid 0–0.01% (W/V) was
used to quantify the total phenolic content.
Statistical analysis
The experiments were designed as a completely ran-
domized design. Statistical analysis was carried out
using SPSS version 16 (SPSS incorporation Chicago,
IL, USA). Duncan’s Multiple Range Tests (P=0.05)
were performed to determine significant differences
among the treatments.
RESULTS AND DISCUSSION
Exocarp browning is the main factor influencing post-
harvest quality and storage life of longan fruits7–9.
It has been reported that the visual appeal of lon-
gan could deteriorate within 3 days under ambient
conditions following harvest4–6. In our study, the
inhibitory effect of SC on exocarp browning and
activities of PPO and POD in longan fruits was in-
vestigated. As shown in Fig. 1, exocarp browning,
represented by a browning index, increased with in-
creasing storage time. The browning symptom was
significantly reduced (p < 0.05) when fruits were
dipped in SC at concentrations of 0.001–0.05% (w/v)
for 10 min and stored at room temperature (25 ±1 °C)
for 48 h (Fig. 1). SC at a concentration of 0.01%
was the most effective treatment in reducing exocarp
browning, reducing by 73.8% and 36.7% at 24 and
48 h, respectively, (Fig. 1). Our results are consistent
with previous studies by Lu et al19,20 and Guan and
Fan22 who found that SC prevented browning of Red
Delicious and Granny Smith apples. In addition,
ClO2, a derivative of SC, has also been reported to
reduce browning of Golden Delicious apples, fresh-
cut lotus roots and fresh-cut asparagus lettuce21,23,24.
Although 0.05% SC was the highest concentration
used in this experiment, the results showed that it
was less effective than 0.01% SC (Fig. 1). A possible
explanation is that the high concentration of 0.05%
SC might cause tissue damage20. Therefore, phenolic
compounds may easily be oxidized by PPO and POD,
resulting in exocarp browning of longan fruits.
As shown in Fig. 2, L* and b* values gradually
decreased with increasing storage time, but dipping
in 0.001–0.05% SC significantly delayed the decrease
in these values, indicating that SC could maintain
lightness and yellowness of longan exocarp. The
exocarp browning of longan fruits is primarily at-
tributed to the oxidation of phenolic compounds by
PPO and POD7,11, leading to rapid browning after
storage at ambient conditions. In our study, changes
a
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0
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5
0
12
24
48
72
Browning index
Storage time (hours)
Control
0.001% SC
0.005% SC
0.01% SC
0.05% SC
Fig. 1 The effects of SC on browning index of longan fruits
during storage at 25 ±1 °C. Bars with the same letters (in
each storage time) are not significantly different at P < 0.05
using LSD. Means and standard errors (n=20).
in PPO and POD activity during storage of longan
are shown in Fig. 3. PPO activity of the control
group dramatically increased and reached the highest
value at 48 h, and this activity gradually decreased at
72 h (Fig. 3a). The result agrees with that of Duan
et al12 who also reported in longan cv. Shixia. All
SC treatments significantly decreased the activity of
PPO as compared to the control group (p < 0.05;
Fig. 3a). The results show that 0.01% and 0.05% SC
significantly reduced PPO activity more than 0.001%
and 0.005% SC treatments at 12–48 h (p < 0.05;
Fig. 3a). The results are consistent with the work of
Lu et al19, Fu et al 21 , and Du et al 23 who reported
that SC and ClO2inhibited PPO activity in fresh-cut
apples and lotus roots. It has been reported that PPO
contains copper in its active site, which is essential for
enzyme activity30,31 . Consequently, SC might affect
the oxidation level of copper and alter the catalysing
activity of PPO32 .
In addition to PPO, POD can catalyse the oxida-
tion of many kinds of phenolic compounds in the pres-
ence of oxygen, which results in enzymatic browning
of harvested fruits such as peach33, litchi 34 , pear35,
and pineapple36. Consequently, the control of POD
activity is important in the preservation of fruits37 .
An increase in POD activity is commonly associated
with injury, flavour loss, or biodegradation38. In our
study, the activity of POD in exocarp of longan fruits
dramatically increased and reached the highest value
at 48 h, and then the activity gradually decreased at
72 h (Fig. 3b). When the fruits were dipped in 0.001–
0.05% SC, the POD activity decreased (Fig. 3b). Our
results show that 0.01% and 0.05% SC significantly
reduced POD activity, with 0.001% and 0.005% being
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ScienceAsia 37 (2011) 237
a
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ab
0
5
10
15
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25
30
35
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12
24
48
72
L* value
Storage time (hours)
Control
0.001% SC
0.005% SC
0.01% SC
0.05% SC
(a)
a
b
b
b
b
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12
24
48
72
b* value
Storage time (hours)
Control
0.001% SC
0.005% SC
0.01% SC
0.05% SC
(b)
Fig. 2 The effects of SC on (a) L* value and (b) b* value of
longan fruits during storage at 25 ±1 °C. Bars with the same
letters (in each storage time) are not significantly different at
p < 0.05 using LSD. Means and standard errors (n=20).
more effective SC treatments (p < 0.05; Fig. 3b).
The underlying mechanism of SC and ClO2treatments
on the inhibitory effect of POD has not been clearly
elucidated24. It is possible that SC might affect the
oxidation level of iron at the active site of POD and
alter the catalysing activity of POD39,40 .
Phenolic compounds are plant secondary metabo-
lites synthesized mostly through the phenylpropanoid
pathway and are involved in the defence of plants
against invading pathogens41 . Various classes of
phenolic compounds such as catechins, catechol hy-
droxycinnamic acid derivatives, and anthocyanins
have been found to contribute to non-enzymatic and
enzymatic browning of foods41. Usually phenolic
compounds in plant organs or tissues are oxidized
into quinones under enzymatic catalysis and then
the quinone is polymerized into brown polymeric
pigments by PPO and POD42. In our study, the
total phenolic content in longan exocarp decreased
dramatically during storage, suggesting that phenolic
compounds were oxidized during the browning pro-
cess (Fig. 4). It was found that dipping in 0.001%-
0.05% SC could significantly delay the decrease in
0
100
200
300
400
500
600
700
0
12
24
48
72
PPO activity (unit/mg protein)
Storage time (hours)
Control
0.001% SC
0.005% SC
0.01% SC
0.05% SC
(a)
0
2,000
4,000
6,000
8,000
10,000
12,000
14,000
0
12
24
48
72
POD activity (unit/mg protein)
Storage time (hours)
Control
0.001% SC
0.005% SC
0.01% SC
0.05% SC
(b)
Fig. 3 The effects of SC on (a) PPO activity and (b) POD
activity of longan fruits during storage at 25 ±1 °C. Means
and standard errors (n=6).
0
200
400
600
800
1,000
1,200
1,400
0
12
24
48
72
Total phenolic content (mg/100g FW)
Storage time (hours)
Control
0.001% SC
0.005% SC
0.01% SC
0.05% SC
Fig. 4 The effects of SC on total phenolic content of longan
fruits during storage at 25 ±1 °C. Means and standard
errors (n=6).
total phenolic content during storage of longan fruits
and the result is compatible with a decrease in the
activity of PPO and POD (Fig. 3).
CONCLUSIONS
The results showed that dipping in 0.001–0.05% SC
for 10 min has the potential to reduce exocarp brown-
ing in longan fruits cv. Daw by reducing the activity
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238 ScienceAsia 37 (2011)
of PPO and POD as well as maintaining total phenolic
content during storage at ambient conditions for 48 h.
It was recommended that an application of SC may be
an alternative method for reducing exocarp browning
and maintaining quality of harvested longan fruits.
Acknowledgements:We would like to thank Dr JF
Maxwell of the Biology Department, Faculty of Science,
Chiang Mai University for helpful reviewing and improving
the manuscript. This study was financially supported by a
grant from the Faculty of Science, Chiang Mai University,
Thailand.
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