Purification of polyphenol oxidase and thebrowning control of litchi fruit by glutathioneand citric acidYueming Jiang,1*† Jiarui Fu,1 Giora Zauberman2 and Yoram Fuchs2

1Department of Biology, Zhongshan University, Guangzhou 510275, PR China2Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, PO Box 6, Bet-Dagan50250, Israel

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 75.6kD by Sephadex G-100 gel ®ltration, and a 108-

fold 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 10mmollÿ1 glutathione

and 100mmolÿ1 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

INTRODUCTIONLitchi fruit (Litchi chinensis Sonn), being native of

subtropical China, deteriorates rapidly once har-

vested, resulting in browning of the pericarp sur-

face.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 bisul-

phites are effective, they can be dangerous to human

health, especially in asthmatic patients.15 Thus alter-

native 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 com-

binations 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 antioxi-

dants (ascorbic acid, L-cysteine, glutathione and n-

propyl 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 METHODSMaterials and treatmentReagents (reduced glutathione, L-cysteine, ascorbic

acid, n-propyl gallate and citric acid) were purchased

from Shanghai Biochemical Co, China. Litchi chinensiscv 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 (20kg) was stored at

ÿ20°C until polyphenol oxidase was extracted and

puri®ed. A second sub-sample (12kg) was dipped in

water containing 10mmolÿ1 l glutathione (reduced

form) and 100mmolÿ1 l citric acid for 5min within 3h

of harvest before being air-dried and packed in units of

20 fruits into polyethylene bags (0.03mm thick),

sealed with a rubber band and stored at 25�2°C for

progressive assessments. We have used the treatment

with 10mmolÿ1 l glutathione and 100mmolÿ1 l citric

acid as this combination showed substantial inhibition

of PPO activity in vitro (Table 1). Fruit dipped for

5min only in water and stored was used as control.

Journal of the Science of Food and Agriculture J Sci Food Agric 79:950±954 (1999)

* 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 ChinaContract/grant sponsor: International Foundation for Science; contract/grant number: E/2265-1Contract/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 assessmentBrowning 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 oxidaseAll steps were carried out at 4°C. The litchi peel was

homogenised with 0.1mol 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 15000�g for

20min, and the supernatant was collected. The

enzyme solution was fractionated with solid ammo-

nium sulphate (50±80% saturation) and the precipi-

tate was collected by centrifugation at 15000�g for

20min, redissoved in 0.01mol lÿ1 sodium phosphate

buffer (pH 7.0) and dialysed against the same buffer.

The dialysed solution was lyophilised, dissolved in

small volume of 0.01mol lÿ1 sodium phosphate buffer

(pH 7.0), and applied to a Sephadex G-200 column

(5.0�110cm), pre-equilibrated with 0.01mol lÿ1 so-

dium 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 0.01mol lÿ1 sodium

phosphate buffer (pH 7.0), the fraction was loaded

onto a column (1.5�36cm) of Q-Sepharose (Fast

Flow) equilibrated with buffer A (0.01mol lÿ1 sodium

phosphate buffer, pH 7.0). The PPO was eluted with a

gradient of 0, 50, 100, 200, 300, 400, 500mol lÿ1

NaCl in buffer A, and the active fraction was pooled.

After ammonium sulphate was added to a ®nal

concentration of 1mol lÿ1, this fraction was loaded

onto a column (1.2�24cm) of Phenyl Sepharose

(Fast Flow) equilibrated with buffer B (0.01mol lÿ1

sodium phosphate, 1mol lÿ1 ammonium sulphate,

1mol lÿ1 KCl, pH 7.0). The PPO was eluted with a

gradient of 100%, 80%, 60%, 40%, 20%, 10% to 0%

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-PAGEanalysis of PPOMolecular weight of the puri®ed enzyme was deter-

mined by Sephadex G-200 ®tration according to the

method of Andrews.20 Cytochrome C (12.5kD),

chymotrypsinogen A (25.0kD), egg albumin

(45.0kD), bovine serum albumin (65.0kD) and g-globulin (125.0kD) 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 3min in Laemmli sample buffer

containing 100mmol 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 determinationPPO activity was assayed with 4-methylcatechol as a

substrate according to spectrophotometric proce-

dure.22 The assay was performed using 0.5ml of

100mmol lÿ1 4-methylcatechol, 1.0ml of 0.1mol lÿ1

sodium phosphate buffer (pH 6.8) and 0.5ml of the

crude enzyme or 1.49ml of 0.1mol lÿ1 sodium

phosphate buffer (pH 6.8) and 0.01ml of the enzyme

solution puri®ed by different steps (see Table 2). The

increase in absorbence at 410nm at 25°C was

recorded automatically for 5min (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 deter-

mined according to the dye-binding method of

Bradford23 with bovine serum protein as the standard.

Effect of various antioxidants on PPO activityA quantity 0.5ml of 100mmol lÿ1 4-methylcatechol,

1.39ml of 0.1mol lÿ1 sodium phosphate buffer (pH

6.8) and 0.1ml of the different concentration solutions

of these antioxidants as given in Table 1, the ®nal

concentration of which was 1mmol lÿ1 or 10mmol lÿ1,

were mixed immediately before the addition of 0.01ml

of the puri®ed enzyme solution. Enzyme activity was

determined using the method described previously

Table 1. Relative inhibitory effects of variousantioxidants with or without 10mmol lÿ1 citric acidon litchi PPO activity

Without citric acid With citric acid

1mmol lÿ1 10mmol lÿ1 1mmol lÿ1 10mmol lÿ1

Glutathione (reduced) 29 11 9 0

L-cysteine 51 23 16 9

Ascorbic acid 89 53 21 17

n-Propyl gallate 85 49 19 13

Citric acid 57 34

A substrate was present at a ®nal concentration of 25mmol lÿ1, and activity was expressed relative to

that with 4-methylcatechol without any chemical inhibitors, which was 1.3� 105 Umgÿ1 protein (=100).

J Sci Food Agric 79:950±954 (1999) 951

Browning control of litchi fruit by glutathione and citric acid

and the relative enzymatic activity was expressed

considering the activity without any chemicals as 100.

Effect of the treatment with glutathione and citricacid on PPO activityPPO activity was measured on 2, 4 and 6 days after

storage at 25°C. Litchi peel (6g) from 6 fruits were

ground with 30ml of 0.1mol lÿ1 sodium phosphate

buffer (pH 6.8) and 0.6g of polyvinglpyrrolidone

(insoluble). After centrifugation at 20000�g for

20min, the supernatant was collected as the crude

enzyme. The assay of the enzyme activity was

performed using 1.0ml of 0.1mol lÿ1 sodium phos-

phate buffer (pH 6.8), 0.5ml of 100mmol lÿ1 4-

methylacatechol, and 0.5ml of the crude enzyme

solution according to the method described above.

Measurements of reduced and oxidised glutathione(GSH and GSSG)Litchi peel (10g) and 30g of pulp from 12 fruits were

homogenised on ice using a Polytron homogeniser.

The solution used for homogenisation consisted of

50ml of 0.1mol lÿ1 sodium phosphate-0.005mol lÿ1

EDTA buffer (pH 8.0) and 10ml of 25% HPO3,

which was used as a protein precipitant. The total

homogenate was centrifuged at 4°C at 100000�g for

30min to obtain the supernatant for the assay of

GSSG and GSH. Determination of GSH was per-

formed by the method of Hissin and Hilf.24 To 0.5ml

of the 100000�g supernatant, 4.5ml of the phos-

phate-EDTA buffer (pH 8.0) was added. The ®nal

assay mixture (2.0ml) contained 0.1ml of the diluted

supernatant, 1.8ml of the phosphate-EDTA buffer,

and 0.1ml of O-phthalaldehyde (OPT) solution,

containing 0.1mg of OPT. After through mixing and

incubation at room temperature for 15min, the

solution was transferred to a quartz cuvette.

Fluorescence at 420nm was determined with the

activation at 350nm using a Perkin-Elmer ¯uores-

cence spectrophotometer (model MPE-3). For GSSG

assay, a 0.5ml portion of the original 100000�gsupernatant was incubated at room temperature with

0.2ml of 0.04mol lÿ1 N-ethylmaleimide (NEM) for

30min to interact with GSH present in the sample. To

this mixture, 4.3ml of 0.1mol lÿ1 NaOH was added. A

0.1ml portion of this mixture was taken for measure-

ment of GSSG, using the procedure outlined above for

GSH assay, except that 0.1mol lÿ1 NaOH was

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 0.1mg, was added. To the second tube, after

homogenisation, 0.1mg 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 analysisEffects of the treatment with 10mmol lÿ1 glutathione

and 100mmol 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 DISCUSSIONPurification and molecular weight estimation of PPOThe 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 75.6kD 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 45.0kD (Fig

1).

Table 2. Purification of polyphenol oxidase of litchi peel

Step (%)

Volume

(ml)

Protein

(mgmlÿ1)

Activity

(Units mlÿ1)

Speci®c activity

(Units mgÿ1 protein)

Total activity

(Units)

Puri®cation

(fold) Yield

Crude extract 15000 10.8 13.2 1.2� 103 198000 1.0 100.0

(NH4)2SO4 250 76.3 259.4 3.4� 103 64850 2.8 32.8

Sephadex G-200 80 7.4 140.6 1.9� 104 11248 15.8 5.7

Q-Sepharose 35 6.2 260.4 4.2� 104 9114 35.0 4.6

Phenyl Sepharose 10 5.8 754.0 1.3� 105 7540 108.3 3.8

Enzyme activity was assayed by using 0.5ml of 100mmol lÿ1 4-methylcatechol and 1.0ml of 0.1mol lÿ1 sodium phosphate buffer (pH 6.8) and 0.5ml of the crude

enzyme or 1.49ml of 0.1mol lÿ1 sodium phosphate buffer (pH 6.8) and 0.01ml of the enzyme solution puri®ed by different steps.

Figure 1. SDS-PAGE of the purified polyphenol oxidase of litchi fruit peel.A, sample; B, standard protein. 1, chymotrypsinogen A (25.0kD); 2, eggalbumin (45.0kD).

952 J Sci Food Agric 79:950±954 (1999)

Y Jiang et al

Chemical inhibition of PPOThe effects of four different antioxidants on the

puri®ed litchi PPO are shown in Table 1. With 4-

methylcatechol 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 (1mmol 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 pro-

ducts.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 (10mmol lÿ1) in combi-

nation with citric acid (10mmol 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

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 Cu-

chelating power.18

Effect of the treatment with glutathione and citricacid on PPO activity and browning of litchi fruitGlutathione 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 (10mmol lÿ1)

in combination with citric acid (10mmol lÿ1) showed

good inhibition of PPO in vitro, it was not very effective

in controlling browning of litchi fruit (data not

shown). When 100mmol lÿ1 of citric acid instead of

10mmol 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 10mmol lÿ1

glutathione and 100mmol lÿ1 citric acid (0.042mg

gÿ1 FW (GSH) and 0.138mggÿ1 FW(GSSG) of peel

and 0.008mggÿ1 FW(GSH) and 0.022mggÿ1 FW

(GSSG) of pulp), and then decreased to 0.018mggÿ1

FW (GSH) and 0.118mggÿ1 FW (GSSG) of peel and

0.003mggÿ1 FW (GSH) and 0.019mggÿ1 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 10mmol lÿ1 glutathione and 100mmol lÿ1

citric acid was markedly higher than that of the fruit

treated with glutathione alone or in combination with

10mmol 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

Table 3. Effect of glutathione(10mmol lÿ1)� citric acid(100mmol lÿ1) on PPO activity andbrowning of litchi fruit

Browning index PPO activity (U mgÿ1 protein)

Days of storage Control Treatment Control Treatment (% of control)

2 1.7a 1.3b 391.7a 60.8b 15

4 2.7a 1.6b 536.4a 92.1b 17

6 4.2a 2.1b 407.2a 81.7b 20

Enzyme activity was assayed by using 0.5ml of 100mmol lÿ1 4-methycatechol, 1.0ml of 0.1mol lÿ1 sodium

phosphate buffer (pH 6.8) and 0.5ml 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.

Figure 2. Molecular weight estimation of litchi polyphenol oxidase bySephadex G-200 gel filtration. V0, void volume of the column; Ve, elutionvolume 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) 953

Browning control of litchi fruit by glutathione and citric acid

higher content of glutathione in litchi peel after the

addition of 100mmol lÿ1 citric acid, resulting in the

delay in browning and better maintenance of the

appearance of litchi fruit.

ACKNOWLEDGEMENTSThe ®nancial support provided in part by the Inter-

national Foundation for Science (E/2265-1), Stock-

holm, Sweden, and MASHAV, Israel, is greatly

appreciated.

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