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Journal of the Science of Food and Agriculture
J Sci Food Agric 79:950±954 (1999)
Purification of polyphenol oxidase and the
browning control of litchi fruit by glutathione
and citric acid
Yueming Jiang,1*† Jiarui Fu,1 Giora Zauberman2 and Yoram Fuchs2
1
Department of Biology, Zhongshan University, Guangzhou 510275, PR China
Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet-Dagan
50250, Israel
2
Abstract: Polyphenol oxidase (PPO, EC 1.10.3.2) was puri®ed to homogeneity from litchi peel yielding
a single protein with a molecular weight of about 75.6 kD by Sephadex G-100 gel ®ltration, and a 108fold puri®cation of PPO achieved. The enzyme was determined to be composed of two similar
subunits. Glutathione, L-cysteine and citric acid suppressed PPO activity markedly, whereas ascorbic
acid and n-propyl gallate showed a little inhibition. Moreover, the effect was enhanced by the addition
of citric acid. On the basis of the inhibition of PPO activity in vitro, the use of 10 mmol l ÿ1 glutathione
and 100 mmolÿ1 l citric acid was found to give good control of the browning of litchi fruit, and an 80±85%
inhibition of PPO activity was observed. It is suggested that application of glutathione in combination
with citric acid may slow down the browning of litchi fruit.
# 1999 Society of Chemical Industry
Keywords: Litchi chinensis Sonn; polyphenol oxidase; puri®cation; browning; glutathione
INTRODUCTION
Litchi fruit (Litchi chinensis Sonn), being native of
subtropical China, deteriorates rapidly once harvested, resulting in browning of the pericarp surface.1±3 Browning has been attributed to a rapid
degradation of the red pigments by polyphenol
oxidase, producing brown colour by-products.4±7
Chemicals can be used to prevent enzymatic
browning such as bisulphite,8,9 ascorbic acid and its
analogies10,11 and cysteine.12±14 Although the bisulphites are effective, they can be dangerous to human
health, especially in asthmatic patients.15 Thus alternative chemicals without toxic effects are needed.
Langdon16 showed that the combination of ascorbic
acid and citric acid prevented enzymatic browning of
sliced potatoes. Santerre et al17 con®rmed that combinations of ascorbic acid, erythorbic acid and citric
acid were ef®cient in preventing browning of sliced
apples. According to Pizzocaro et al18 sliced apples
could be protected from browning using a mixture of
ascorbic acid and calcium chloride. In the present
work, the litchi PPO was ®rst extracted and puri®ed,
and then the inhibiting effects of these four antioxidants (ascorbic acid, L-cysteine, glutathione and npropyl gallate), used alone or in combination with
citric acid, were studied. The best combination of
glutathione and citric acid was adopted to examine its
effect on the control of the browning of litchi fruit.
MATERIALS AND METHODS
Materials and treatment
Reagents (reduced glutathione, L-cysteine, ascorbic
acid, n-propyl gallate and citric acid) were purchased
from Shanghai Biochemical Co, China. Litchi chinensis
cv Huaizhi (a major cultivar in China) was obtained
from a commercial orchard in Guangzhou, PR China.
Fruits were selected for uniformity of shape, colour
and size, and any blemished or diseased fruits
discarded. A sub-sample of fruit (20 kg) was stored at
ÿ20 °C until polyphenol oxidase was extracted and
puri®ed. A second sub-sample (12 kg) was dipped in
water containing 10 mmolÿ1 l glutathione (reduced
form) and 100 mmolÿ1 l citric acid for 5 min within 3 h
of harvest before being air-dried and packed in units of
20 fruits into polyethylene bags (0.03 mm thick),
sealed with a rubber band and stored at 25 2 °C for
progressive assessments. We have used the treatment
with 10 mmolÿ1 l glutathione and 100 mmolÿ1 l citric
acid as this combination showed substantial inhibition
of PPO activity in vitro (Table 1). Fruit dipped for
5 min only in water and stored was used as control.
* Correspondence to: Dr Yueming Jiang, Department of Biology, Zhongshan University, Guangzhou 510275, PR China
†
Current address: Dr Yueming Jiang, South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650, PR China
Contract/grant sponsor: International Foundation for Science; contract/grant number: E/2265-1
Contract/grant sponsor: MASHAV
(Received 29 September 1997; accepted 28 August 1998)
# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50
950
Browning control of litchi fruit by glutathione and citric acid
Without citric acid
Table 1. Relative inhibitory effects of various
antioxidants with or without 10mmol lÿ1 citric acid
on litchi PPO activity
Glutathione (reduced)
L-cysteine
Ascorbic acid
n-Propyl gallate
Citric acid
1 mmol lÿ1
10 mmol lÿ1
1 mmol lÿ1
10 mmol lÿ1
29
51
89
85
57
11
23
53
49
34
9
16
21
19
0
9
17
13
A substrate was present at a ®nal concentration of 25 mmol lÿ1, and activity was expressed relative to
that with 4-methylcatechol without any chemical inhibitors, which was 1.3 105 U mgÿ1 protein (=100).
Browning assessment
Browning was estimated by measuring the extent of
the total browned area on each fruit pericarp on the
following scale:19 1, no browning (excellent quality);
2, slight browning; 3, <1/4 browning; 4, 1/4±1/2
browning; 5, >1/2 browning (poor quality). The
browning index was calculated using the following
formula:
X
(browning scale percentage of corresponding
fruit within each class)
Fruit evaluated at a higher index than 3.0 was
considered to be unacceptable for marketing.
Extraction and purification of polyphenol oxidase
All steps were carried out at 4 °C. The litchi peel was
homogenised with 0.1 mol lÿ1 sodium phosphate
buffer (pH 7.0) using an Ultra-AII (Guangdong,
China). After ®ltration of the homogenate through a
muslin, the ®ltrate was centrifuged at 15 000 g for
20 min, and the supernatant was collected. The
enzyme solution was fractionated with solid ammonium sulphate (50±80% saturation) and the precipitate was collected by centrifugation at 15 000 g for
20 min, redissoved in 0.01 mol lÿ1 sodium phosphate
buffer (pH 7.0) and dialysed against the same buffer.
The dialysed solution was lyophilised, dissolved in
small volume of 0.01 mol lÿ1 sodium phosphate buffer
(pH 7.0), and applied to a Sephadex G-200 column
(5.0 110 cm), pre-equilibrated with 0.01 mol lÿ1 sodium phosphate buffer (pH 7.0). The enzyme solution
was eluted with the same buffer and the fraction with
highest enzymatic activity was pooled and lyophilised.
Dissolved in small volume of 0.01 mol lÿ1 sodium
phosphate buffer (pH 7.0), the fraction was loaded
onto a column (1.5 36 cm) of Q-Sepharose (Fast
Flow) equilibrated with buffer A (0.01 mol lÿ1 sodium
phosphate buffer, pH 7.0). The PPO was eluted with a
gradient of 0, 50, 100, 200, 300, 400, 500 mol lÿ1
NaCl in buffer A, and the active fraction was pooled.
After ammonium sulphate was added to a ®nal
concentration of 1 mol lÿ1, this fraction was loaded
onto a column (1.2 24 cm) of Phenyl Sepharose
(Fast Flow) equilibrated with buffer B (0.01 mol lÿ1
sodium phosphate, 1 mol lÿ1 ammonium sulphate,
1 mol lÿ1 KCl, pH 7.0). The PPO was eluted with a
gradient of 100%, 80%, 60%, 40%, 20%, 10% to 0%
J Sci Food Agric 79:950±954 (1999)
With citric acid
buffer B in buffer A. The active fraction from the
Phenyl Sepharose column was pooled and lyophilised,
dissolved in a small volume of buffer A. After overnight
dialysis against the same buffer, the dialysed solution
was collected as the puri®ed enzyme.
Molecular weight estimation and SDS-PAGE
analysis of PPO
Molecular weight of the puri®ed enzyme was determined by Sephadex G-200 ®tration according to the
method of Andrews.20 Cytochrome C (12.5 kD),
chymotrypsinogen A (25.0 kD), egg albumin
(45.0 kD), bovine serum albumin (65.0 kD) and gglobulin (125.0 kD) were used as marker proteins.
Electrophoresis of the enzyme was carried out using
the gel system of Laemmli.21 Fully denatured samples
were boiled for 3 min in Laemmli sample buffer
containing 100 mmol lÿ1 DTT as a reducing agent
and 2% SDS. Gels were stained with Coomassie blue
to detect protein, and the molecular weight of PPO
was determined by comparison with known standards.
Enzyme assay and protein determination
PPO activity was assayed with 4-methylcatechol as a
substrate according to spectrophotometric procedure.22 The assay was performed using 0.5 ml of
100 mmol lÿ1 4-methylcatechol, 1.0 ml of 0.1 mol lÿ1
sodium phosphate buffer (pH 6.8) and 0.5 ml of the
crude enzyme or 1.49 ml of 0.1 mol lÿ1 sodium
phosphate buffer (pH 6.8) and 0.01 ml of the enzyme
solution puri®ed by different steps (see Table 2). The
increase in absorbence at 410 nm at 25 °C was
recorded automatically for 5 min (Beckman, DU-7).
One unit of enzyme activity was de®ned as the amount
of the enzyme which caused a change of 0.001 in
absorbence per minute. Protein content was determined according to the dye-binding method of
Bradford23 with bovine serum protein as the standard.
Effect of various antioxidants on PPO activity
A quantity 0.5 ml of 100 mmol lÿ1 4-methylcatechol,
1.39 ml of 0.1 mol lÿ1 sodium phosphate buffer (pH
6.8) and 0.1 ml of the different concentration solutions
of these antioxidants as given in Table 1, the ®nal
concentration of which was 1 mmol lÿ1 or 10 mmol lÿ1,
were mixed immediately before the addition of 0.01 ml
of the puri®ed enzyme solution. Enzyme activity was
determined using the method described previously
951
Y Jiang et al
Table 2. Purification of polyphenol oxidase of litchi peel
Step (%)
Crude extract
(NH4)2SO4
Sephadex G-200
Q-Sepharose
Phenyl Sepharose
Volume
(ml)
15 000
250
80
35
10
Protein
(mg mlÿ1)
10.8
76.3
7.4
6.2
5.8
Activity
(Units mlÿ1)
13.2
259.4
140.6
260.4
754.0
Speci®c activity
(Units mgÿ1 protein)
Total activity
(Units)
Puri®cation
(fold)
Yield
198 000
64 850
11 248
9114
7540
1.0
2.8
15.8
35.0
108.3
100.0
32.8
5.7
4.6
3.8
3
1.2 10
3.4 103
1.9 104
4.2 104
1.3 105
Enzyme activity was assayed by using 0.5 ml of 100 mmol lÿ1 4-methylcatechol and 1.0 ml of 0.1 mol lÿ1 sodium phosphate buffer (pH 6.8) and 0.5 ml of the crude
enzyme or 1.49 ml of 0.1 mol lÿ1 sodium phosphate buffer (pH 6.8) and 0.01 ml of the enzyme solution puri®ed by different steps.
and the relative enzymatic activity was expressed
considering the activity without any chemicals as 100.
Effect of the treatment with glutathione and citric
acid on PPO activity
PPO activity was measured on 2, 4 and 6 days after
storage at 25 °C. Litchi peel (6 g) from 6 fruits were
ground with 30 ml of 0.1 mol lÿ1 sodium phosphate
buffer (pH 6.8) and 0.6 g of polyvinglpyrrolidone
(insoluble). After centrifugation at 20 000 g for
20 min, the supernatant was collected as the crude
enzyme. The assay of the enzyme activity was
performed using 1.0 ml of 0.1 mol lÿ1 sodium phosphate buffer (pH 6.8), 0.5 ml of 100 mmol lÿ1 4methylacatechol, and 0.5 ml of the crude enzyme
solution according to the method described above.
Measurements of reduced and oxidised glutathione
(GSH and GSSG)
Litchi peel (10 g) and 30 g of pulp from 12 fruits were
homogenised on ice using a Polytron homogeniser.
The solution used for homogenisation consisted of
50 ml of 0.1 mol lÿ1 sodium phosphate-0.005 mol lÿ1
EDTA buffer (pH 8.0) and 10 ml of 25% HPO3,
which was used as a protein precipitant. The total
homogenate was centrifuged at 4 °C at 100 000 g for
30 min to obtain the supernatant for the assay of
GSSG and GSH. Determination of GSH was performed by the method of Hissin and Hilf.24 To 0.5 ml
of the 100 000 g supernatant, 4.5 ml of the phosphate-EDTA buffer (pH 8.0) was added. The ®nal
assay mixture (2.0 ml) contained 0.1 ml of the diluted
supernatant, 1.8 ml of the phosphate-EDTA buffer,
and 0.1 ml of O-phthalaldehyde (OPT) solution,
containing 0.1 mg of OPT. After through mixing and
incubation at room temperature for 15 min, the
solution was transferred to a quartz cuvette.
Fluorescence at 420 nm was determined with the
activation at 350 nm using a Perkin-Elmer ¯uorescence spectrophotometer (model MPE-3). For GSSG
assay, a 0.5 ml portion of the original 100 000 g
supernatant was incubated at room temperature with
0.2 ml of 0.04 mol lÿ1 N-ethylmaleimide (NEM) for
30 min to interact with GSH present in the sample. To
this mixture, 4.3 ml of 0.1 mol lÿ1 NaOH was added. A
0.1 ml portion of this mixture was taken for measurement of GSSG, using the procedure outlined above for
GSH assay, except that 0.1 mol lÿ1 NaOH was
952
employed as diluent rather than phosphate-EDTA
buffer. The recovery of GSH was estimated in the
following way. Equal amounts of peel or pulp were
homogenised in three different tubes. In the ®rst tube,
prior to homogenisation, a known amount of GSH,
usually 0.1 mg, was added. To the second tube, after
homogenisation, 0.1 mg of GSH was added. The third
tube acted as the control and no addition of GSH was
made prior to or after homogenisation. The three
homogenates were subsequently handled identically
for GSH measurement. Estimation of GSSG recovery
was performed in a similar manner.
Statistical analysis
Effects of the treatment with 10 mmol lÿ1 glutathione
and 100 mmol lÿ1 citric acid on PPO activity and
browning of post-harvest litchi fruit were repeated
three times with similar results and data were analysed
using Duncan's multiple range test (<0.05).
RESULTS AND DISCUSSION
Purification and molecular weight estimation of PPO
The puri®ed PPO was homogenous as judged by the
single band produced on the SDS-PAGE, and a
summary of typical puri®cation of the enzyme is given
in Table 2. The molecular weight of PPO was
estimated to be about 75.6 kD using gel ®ltration
(Fig 2), and the enzyme determined to be composed of
two similar subunits, based on the results showing that
the molecular mass of the protein band of the enzyme
on the SDS-PAGE was between 25.0 and 45.0 kD (Fig
1).
Figure 1. SDS-PAGE of the purified polyphenol oxidase of litchi fruit peel.
A, sample; B, standard protein. 1, chymotrypsinogen A (25.0 kD); 2, egg
albumin (45.0 kD).
J Sci Food Agric 79:950±954 (1999)
Browning control of litchi fruit by glutathione and citric acid
Browning index
Days of storage
2
4
6
Table 3. Effect of glutathione
(10 mmol lÿ1) ‡ citric acid
(100mmol lÿ1) on PPO activity and
browning of litchi fruit
Control
Treatment
Control
Treatment
(% of control)
1.7a
2.7a
4.2a
1.3b
1.6b
2.1b
391.7a
536.4a
407.2a
60.8b
92.1b
81.7b
15
17
20
Enzyme activity was assayed by using 0.5 ml of 100 mmol lÿ1 4-methycatechol, 1.0 ml of 0.1 mol lÿ1 sodium
phosphate buffer (pH 6.8) and 0.5 ml of the crude enzyme (pH 6.8).
Corresponding means within a line between control and treatment followed by the same letter are not signi®cantly
different at the 5% level.
Chemical inhibition of PPO
The effects of four different antioxidants on the
puri®ed litchi PPO are shown in Table 1. With 4methylcatechol as a substrate, lag periods were
observed when glutathione, L-cysteine, ascorbic acid
and n-propyl gallate were used. The inhibition of PPO
activity was enhanced with increasing concentration of
the antioxidants in the solutions. Of these four
antioxidants used in this study, glutathione was the
best, followed by L-cysteine. Ascorbic acid or n-propyl
gallate (1 mmol lÿ1) showed a little inhibition of PPO.
Cysteine can easily form complexes with quinones and
PPO was inhibited by the formation of additional
products.25,26 This amino acid has been suggested to
prevent enzymatic browning in processed fruit products.12 Janovitz-Klapp et al26 reported that, using
Red Delicious apple PPO, the higher the cysteine
concentration, the longer the lag period and the lower
the rate of browning following the lag period.
The inhibiting effect of PPO using these four
antioxidants was enhanced by the addition of citric
acid (Table 1). Glutathione (10 mmol lÿ1) in combination with citric acid (10 mmol lÿ1) suppressed the
PPO activity completely. The ®nal pH of the reaction
mixture, however, was not affected markedly after the
addition of citric acid and still was maintained between
6.3 and 6.5. Jiang et al7 pointed out that litchi PPO
Figure 2. Molecular weight estimation of litchi polyphenol oxidase by
Sephadex G-200 gel filtration. V0, void volume of the column; Ve, elution
volume of the substances; MW, molecular weight in kD. 1, g-globulin; 2,
bovine serum albumin; 3, egg albumin; 4, chymotrysinogen A; 5,
cytochrome C; A, purified enzyme.
J Sci Food Agric 79:950±954 (1999)
PPO activity (U mgÿ1 protein)
showed good activity between pH 6.0 and pH 7.5 with
4-methylcatechol as a substrate, optimum pH being
6.8. Citric acid is not an antioxidant agent, this its
inhibiting effect could be related to the phenolase Cuchelating power.18
Effect of the treatment with glutathione and citric
acid on PPO activity and browning of litchi fruit
Glutathione was adopted to carry out post-harvest
treatment of litchi fruit, as glutathione inhibited
markedly PPO activity in vitro (Table 1), and it is safe
to human health. Although glutathione (10 mmol lÿ1)
in combination with citric acid (10 mmol lÿ1) showed
good inhibition of PPO in vitro, it was not very effective
in controlling browning of litchi fruit (data not
shown). When 100 mmol lÿ1 of citric acid instead of
10 mmol lÿ1 was used, the PPO inhibition increased to
80% and the browning index decreased to 2.1,
whereas the control was higher than 4.0 at 25 2 °C,
6 days after storage (Table 3). The treatment was
ef®cient, as the treated fruit remained bright red in
colour, while the initial red colour of the control fruit
had largely disappeared by the end of 6-day storage at
25 °C. Browning has been studied in other fruit, and
discoloration correlated well with PPO activity and
phenolic concentration.27,28 Consequently, resulting
brown pigmentation has attributed directly to PPO
action.29
Glutathione residue in the treated fruit was highest
immediately after the treatment with 10 mmol lÿ1
glutathione and 100 mmol lÿ1 citric acid (0.042 mg
gÿ1 FW (GSH) and 0.138 mg gÿ1 FW(GSSG) of peel
and 0.008 mg gÿ1 FW(GSH) and 0.022 mg gÿ1 FW
(GSSG) of pulp), and then decreased to 0.018 mg gÿ1
FW (GSH) and 0.118 mg gÿ1 FW (GSSG) of peel and
0.003 mg gÿ1 FW (GSH) and 0.019 mg gÿ1 FW
(GSSG) of pulp, respectively. Most of the residue
was located in the non-edible skin. In addition, it was
observed that the content of glutathione of the fruit
treated with 10 mmol lÿ1 glutathione and 100 mmol lÿ1
citric acid was markedly higher than that of the fruit
treated with glutathione alone or in combination with
10 mmol lÿ1 citric acid, especially in the content of
GSH in litchi peel (data not shown). Among these
treatments, the content of glutathione of the fruit
treated only with glutathione was the lowest. These
differences may suggest that the PPO inhibition was
attributed not only to acid nature, but also to the
953
Y Jiang et al
higher content of glutathione in litchi peel after the
addition of 100 mmol lÿ1 citric acid, resulting in the
delay in browning and better maintenance of the
appearance of litchi fruit.
ACKNOWLEDGEMENTS
The ®nancial support provided in part by the International Foundation for Science (E/2265-1), Stockholm, Sweden, and MASHAV, Israel, is greatly
appreciated.
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J Sci Food Agric 79:950±954 (1999)
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