Effects of Hydrocooling and Chitosan Coating on Browning and

Physiological Changes in Fresh-cut Rose Apple

W. Worakeeratikul, V. Srilaong, A. Uthairatanakij and P. Jitareerat
Division of Postharvest Technology
School of Bioresources and Technology
King Mongkut’s University of Technology Thonburi
Bangkhuntien Campus
Bangkok 10150
Thailand

Keywords: browning, chitosan, fresh-cut rose apple, physiology

Abstract
Effects of chitosan coating on browning and physiological changes in fresh-
cut rose apple were studied. Cooled fruit were cut into four sections and treated with
chitosan (4°C) at concentrations of 0 (control), 0.05, 0.1 and 0.2% (v/v), wrapped
with PVC film and stored at 5°C for 72 h. Chitosan retarded browning, maintained
L value, total soluble solids, reducing sugar content and titratable acidity compared
to untreated sections. Chitosan coating had no significant effects on the changes of
flesh colour (b value), phenolic content, polyphenol oxidase (PPO) activity, flesh
texture, and CO2 and O2 gas in the package. Chitosan coating increased CO2
concentration in the section of fresh-cut rose apple.

INTRODUCTION
Fresh-cut fruit are fresh raw fruit sold ready-to-eat that are usually trimmed,
peeled, washed, dried, cut, and packed in sealed pouches or in trays wrapped with
extensible film (Baldwin et al., 1996). The practices of fresh-cut processing result in
quality deterioration associated with water loss, softening, microbial contamination,
increased respiration and cut surface browning and they shorten shelf life (Guerzoni et al.,
1996; Watada et al., 1996).
Browning of cut surface is a major problem of fresh-cut rose apple. Browning of
fresh-cut produces is due to oxidative reactions of phenolic compounds by
polyphenoloxidase and the reaction products, o-quinones, to various polymerized
products (Ahvenainen, 2000). The use of edible coating has been found to be effective in
reducing browning and decay of fresh-cut (Ahvenainen, 1996). Edible coatings can offer
a possibility to extend the shelf life of fresh-cut products by providing a semipermeable
barrier to gases and water vapor, and thereby reducing respiration, oxidative reactions and
water loss (Baldwin et al., 1995). Chitosan is a polycationic biopolymer industrially
produced by chemical de-acetylation of chitin which is found in arthropod exoskeletons
(Durango et al., 2006). Several authors have reported beneficial effects of this biopolymer
as a coating on products including longan (Jiang and Li, 2001), strawberry (Han et al.,
2004; Hernández-Muñoz et al., 2006), fresh-cut Chinese water chestnut (Pen and Jiang,
2003) and fresh-cut mango (Chien et al., 2007). Chitosan coatings have the potential to
retard water loss, colour changes, respiration rate and extend shelf life of strawberry,
longan, litchi and fresh-cut mango (El Ghaouth et al., 1991; Jiang and Li, 2001; Jiang et
al., 2005; Chien et al., 2007).
The purpose of this research was to study the effects of chitosan coating on
browning and physiological changes of fresh-cut rose apple during storage at low
temperature.

MATERIALS AND METHODS

Plant Material Preparation and Chitosan Treatments

Rose apple (Eugenia jumbos ‘Tup Tim Jun’) fruit at full maturity (45 days after

Proc. IC on Qual. Manag. Fresh Cut Produce 427

Eds.: S. Kanlayanarat et al.
Acta Hort. 746, ISHS 2007
full bloom) were harvested from an orchard in Nakornpathom Province, Thailand. The
fruit were transported to postharvest technology laboratory. The fruit were pre-cooled in
50 mg L-1 chlorinated water at 4°C to a core temperature of 13°C and then cut into four
sections. The rose apple sections were treated with a 4°C chitosan solution (Bio Safer Co.,
Ltd., Thailand) at concentrations of 0 (distilled water as control), 0.05, 0.10 and 0.20%
(v/v). Eight treated sections were placed on the foam tray and wrapped with PVC film
before being stored at 5°C for 72 h.

Measurements of Quality Changes

Colour measurement was done with a colorimeter (Minolta DP-301 model, Japan).
L (lightness) and b (yellow-blue) were determined. The measurements were done at the
cut surface of rose apple sections at 0, 6, 12, 24, 48 and 72 h of shelf life. Browning at the
cut surface of rose apple sections was evaluated using a visual hedonic scale; where 1 =
none, 2 = slight brown, 3 = moderate brown, 4 = severe brown and 5 = extremely severe
brown. Browning score was made at 0, 6, 12, 18, 24, 48 and 72 h of shelf life.
Weight was monitored every 24 h and weight loss was calculated as a percentage
of the initial weight. Flesh texture of fresh-cut rose apple was assessed every 24 h using a
texture analyser (TA-XT2, England) and expressed as shear force in N. A shear test was
performed using a 7x12 cm stainless knife of 3 mm thickness and a head speed of 1.5
cm/min. 3 g of fresh-cut rose apple was homogenized in a blender and the homogenate
was used to measure TA and TSS. TA (citric acid equivalent) was measured by titrating
the homogenate to the end point of 0.1 M NaOH. The TSS was measured by using a hand
refractometer (ATAGO, Model N1). The TSS was recorded after calibration and shown
as ºBrix.
Total phenolics were measured with the method of Chaiprasart et al. (2001). Flesh
(5 g) was homogenized in 10 ml of 80% ethanol and centrifuged at 12000 × g for 15 min
at room temperature. One ml of supernatant was diluted with 9 ml distilled water. One ml
of diluted supernatant was mixed with 5 ml of 1 N Folin-Ciocaltou reagent and 4 ml of
2.5 M Na2CO3. The mixture was heated at 30°C for 1 hour, and cooled at 0°C for 1 hour.
The absorbance was measured at 765 nm with a spectrophotometer (UV-1601, Japan) and
compared with the standard curve of gallic acid. The concentration of phenolic was
calculated as g/100 g fresh weight.
PPO activity was measured according to the method of Lichter et al. (2000). A
flesh sample (2 g) was homogenized in 20 ml of 0.1 M sodium phosphate buffer (pH 6.6)
and 0.5 g of polyvinyl pyrollidone. The homogenate was centrifuged at 8000 × g for 10
min at 4°C. The supernatant was collected and centrifuged again at 10000 × g for 20 min
at 4°C. The supernatant was collected into a fresh tube and used for the PPO assay. The
assay of PPO activity was performed using 0.12 ml of 23 mM 4-methyl catechol and 0.75
ml of the crude enzyme sample. The mixture was measured with spectrophotometer at
410 nm and linear progress of the reaction was recorded between 10 and 30 sec. The PPO
activity was calculated as ΔOD410/mg protein·min. The protein content was determined
according to the dye-binding method of Bradford (1976) with albumin bovine serum as
the standard protein.
Analysis of O2 and CO2 concentrations in the package during storage was
measured by head space oxygen/carbon dioxide analyzer (Illinois 6600). CO2
concentrations from the internal sections of fresh-cut rose apple were measured according
to the method of Momen et al. (1997).
Reducing sugars were measured according to the method of Hodge and Hofreiter
(1962). The fresh-cut rose apple tissue was vacuum freeze dried (FD-1, Japan). The dry
sample was ground to powder and 0.025 g of sample was placed into a 50 ml flask and 10
ml of 50% ethanol was added. The mixture was incubated in a hot air oven at 60°C for 2
h, shaking every 30 min, and then cooled to room temperature. The mixture was filtered
through Whatman No.42 paper and distilled water was added to make a volume up to 50
ml. One ml of the diluted supernatant was mixed with 1 ml alkaline copper reagent and
heated in boiling water for 15 min before cooling down in a cooling bath. One ml of

428
arsenomolybdic reagent was added and mixed by vortex. The mixture was adjusted with
distilled water to 12.5 ml and incubated at room temperature for 30 min. The mixture
sample was determined by a spectrophotometer at 500 nm with D-glucose as the standard
curve. Unit of reducing sugars was expressed as mg/g dry weight.
There were three replicates per treatment. All data were tested by analysis of
variance (ANOVA procedure). The treatment means were separated using the Duncan’s
multiple range test (DMRT) method. Differences at p≤0.05 were considered as
significant.

RESULTS AND DISCUSSION

Figure 1A and B show the changes of L and b value in fresh-cut rose apple treated
with chitosan after being stored for 72 h at 5°C. L value of uncoated and chitosan coated
fresh-cut rose apple rapidly decreased within 12 h of storage, consequently slowly
declined during 24-72 h. At 12 h, there were no significant differences among the
treatments. However, L value of fresh-cut rose apple treated with chitosan at all
concentrations was significantly higher compared to uncoated during storage for 24-72 h.
This result suggests that chitosan coatings prevent changes of L value. There were no
significant differences in b value of fresh-cut rose apple between treatments. Chitosan
coatings delayed browning in fresh-cut rose apple in comparison with non-coating (Fig.
1C). Browning of the uncoated fresh-cut rose apple appeared at 6 h of storage time while
in the fresh-cut rose apple coated with chitosan at 0.05, 0.1 and 0.2% browning appeared
at 24 h. The data show that browning score of uncoated was significantly higher than of
chitosan coated fresh-cut rose apple. It has been reported that chitosan coatings reduce
browning and maintain L value in fresh-cut Chinese water chestnut (Pen and Jiang, 2003)
and fresh-cut mango (Chien et al., 2007).
Browning in fresh produce is caused by the activity of enzymes associated with
browning such as PPO (Soliva-Fortuny and Martín-Belloso, 2003). In this experiment, the
increase of browning was generally related with the increase of PPO activity during
storage for 72 h (Fig. 1D). However, chitosan coatings did not delay PPO activity in
fresh-cut rose apple because the activities of PPO in chitosan coatings and non-coating
were not significantly different. This is in contrast to Pen and Jiang (2003) who showed
that the PPO activity was inhibited by 2.0% chitosan coating in fresh-cut Chinese water
chestnut. Accumulation of phenolic compounds was observed in fresh-cut rose apple
during storage and there were no significant differences in all treatments (Fig. 1E).
At 72 h of storage, the lowest weight loss was found in 0.2% chitosan coated
fresh-cut rose apple followed by 0.05 and 0.10% chitosan coatings, while weight loss of
uncoated fresh-cut rose apple was highest (Fig. 2A). Although chitosan coatings retarded
weight loss of fresh-cut rose apple, the differences were not significant. Other research
showed that 2.0% chitosan could prevent water loss better than 0.5 and 1.0% chitosan in
longan fruit (Jiang and Li, 2001) and fresh-cut mango (Chien et al., 2007).
There were no significant differences in shear force between treatments 72 h of
storage (Fig. 2B). This suggests that chitosan coating cannot maintain flesh texture of
fresh-cut rose apple.
TSS of fresh-cut rose apple uncoated and coated with chitosan significantly
increased throughout 72 h of storage. TSS of fresh-cut rose apple coated with chitosan
was lower than control. At 72 h, TSS of fresh-cut rose apple coated with 0.2% chitosan
(7.5°Brix) was significantly lower compared to all other treatments (8.5-9.45°Brix),
whereas TSS of fresh-cut rose apple coated with chitosan at 0, 0.05 and 0.1% showed no
significant differences (Fig. 2C). This is in contrast with the result of Chien et al. (2007)
who found that TSS of chitosan coated fresh-cut mango was higher than non-coated.
There was a decrease in TA throughout the storage time and there were no
significant differences among treatments during 0-48 h of storage, except that the TA of
uncoated fruit was significantly less than of chitosan coated fruit at the end of storage (Fig.
2D). This result suggests that chitosan coating can maintain the changes of TA in fresh-
cut rose apple.

429
 The changes of O2 and CO2 in package of fresh-cut rose apple coated with
chitosan are shown in Figure 3. The O2 concentrations in the package of all treatments
decreased throughout storage time while CO2 concentrations increased until 24 h and
remained stable thereafter. At 72 h, the concentrations of both gases in chitosan coated
and uncoated fresh-cut rose apple were not significantly different.
The internal CO2 levels of rose apple sections coated with 0.05, 0.1 and 0.2%
chitosan increased throughout storage whereas level of non-coated rose apple sections
was stable (Fig. 4). At 24-72 h, the internal CO2 levels of chitosan coated rose apple
sections were significantly higher than that of control. After 72 h, the internal CO2
accumulation of rose apple sections coated with 0.1 and 0.2% chitosan significantly
increased when compared to 0.05% chitosan and uncoated. Despite high CO2 levels, off-
flavor was not detected in fresh-cut rose apple when chitosan at 0.05-0.2% was used.
Jianmin et al. (1998) found that ‘Janagold’ apple fruit coated with chitosan caused
accumulation of CO2 in the fruit.
Reducing sugars of chitosan coated and uncoated fresh-cut rose apple increased
throughout storage. Reducing sugars of fresh-cut rose apple coated with 0.2% chitosan
were significantly lower than in other treatments except at 48 h of storage. It seems that
chitosan coating could delay the increase of reducing sugars (Fig. 5).

CONCLUSIONS
Chitosan coatings decreased browning, delayed TSS, reducing sugars and TA of
fresh-cut rose apple compared to non-coating. But chitosan coatings didn’t show
significant effects to reduce phenolic content, PPO activity, flesh texture, and CO2 and O2
in package of fresh-cut rose apple. Moreover, chitosan coatings at 0.05-0.2% caused the
accumulation of internal CO2 but the levels of internal CO2 did not affect flavor in fresh-
cut rose apple.

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Figures

74 A 25 B
73
72 20
71
70
L value

15

b value
69
68 10
67
66 5
65
64 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Storage time (hours) Storage time (hours)

6 10
C (ΔOD410/min/mg protein) D
5 8
Browning (score)

PPO activity

4 6
3
4
2
1 2

0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Storage time (hours) Storage time (hours)

5
E
4.5
Total phenolic compounds

4
3.5
(g/100 g FW)

3
2.5
2
1.5
1
0.5
0
0 12 24 36 48 60 72
Storage time (hours)

Control 0.05% Chi 0.10% Chi 0.20% Chi

Fig. 1. Effects of chitosan coatings at the concentrations of 0 (control), 0.05, 0.1 and
0.2% on L value (A), b value (B), browning changes (C), PPO activity (D) and
total phenolics (E) of fresh-cut rose apple at 5°C over 72 h. Vertical bars represent
standard error of the mean of three replications.

432
 0.3 180
A B
160
0.25 140

Shear force (N)

Weight loss (%)

0.2 120
100
0.15 80
0.1 60
40
0.05
20
0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Storage time (hours) Storage time (hours)

12 C 0.25 D
Total soluble solids (°Brix)

Titratable acidity (% )
10 0.2
8
0.15
6
0.1
4
2 0.05
0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Storage time (hours)
Storage time (hours)

Control 0.05% Chi 0.10% Chi 0.20% Chi

Fig. 2. Effect of chitosan coatings at the concentrations of 0 (control), 0.05, 0.1 and 0.2%
on weight loss (A), shear force (B), total soluble solids (C) and titratable acidity
(D) of fresh-cut rose apple at 5°C for 72 h. Vertical bars represent standard error
of the mean of three replications.

25 A 5 B
CO 2 in package (% )
O2 in package (%)

20 4
15 3
10 2
5 1
0 0
0 12 24 36 48 60 72 0 12 24 36 48 60 72
Storage time (hours) Storage time (hours)

Control 0.05% Chi 0.10% Chi 0.20% Chi

Fig. 3. Effect of chitosan (Chi) coatings at the concentrations of 0 (control), 0.05, 0.1 and
0.2% on O2 (A) and CO2 (B) in package of fresh-cut rose apple at 5°C for 72 h.
Vertical bars represent standard error of the mean of three replications.

433
 0.6
0.5

Internal CO2 (%)

0.4
0.3
0.2
0.1
0
0 12 24 36 48 60 72
Storage time (hours)

Control 0.05% Chi 0.10% Chi 0.20% Chi

Fig. 4. Effect of chitosan coatings at the concentrations of 0 (control), 0.05, 0.1 and 0.2%
on internal CO2 accumulation of fresh-cut rose apple at 5°C for 72 h. Vertical bars
represent standard error of the mean of three replications.

3.5
3
(mg/g dry weight)
Reducing sugars

2.5
2
1.5
1
0.5
0
0 12 24 36 48 60 72
Storage time (hours)

Control 0.05% Chi 0.10% Chi 0.20% Chi

Fig. 5. Effect of chitosan coatings at the concentrations of 0 (control), 0.05, 0.1 and 0.2%
on reducing sugar content of fresh-cut rose apple at 5°C for 72 h. Vertical bars
represent standard error of the mean of three replications.

434