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Int. J. Chem. Sci.: 8(5), 2010, S550-S558
________________________________________
*
Author for correspondence; E-mail: bidyut2006@gmail.com
INHIBITION OF POLYPHENOL OXIDASE IN BANANA,
APPLE AND MUSHROOM BY USING DIFFERENT ANTI-
BROWNING AGENTS UNDER DIFFERENT CONDITIONS
SAMANTA ARPITA, DAS SUBROTO, BHATTACHARYYA PINAKI and
BANDYOPADHYAY BIDYUT
*
Post Graduate Department of Biotechnology, Oriental Institute of Science and Technology, Dewandighi,
Mirjapur, BURDWAN – 02 (W.B.) INDIA
ABSTRACT
Fruit are highly perishable and maintain an active metabolism during the postharvest phase.
Polyphenol oxidase or PPO (EC 1.14.18.1) is considered the enzyme responsible for quality deterioration
and browning in different fruits during postharvest period. Since enzymatic browning causes deterioration
of sensory and nutritional quality and affects appearance and organoleptic properties, inactivation of PPO
is desirable for preservation of foods. The objective of this study was to evaluate the antibrowning
(inhibition of polyphenol oxidase activity) effect of Cysteine (Cys) , Ascorbic acid (AA), citric acid (CA),
sodium metabisulphite(SMB) alone or in combination, at three different pH (3.5,4 and 4.5) in banana
(Musa paradisiaca L. var. Kanthali), apple (Malus pumila Mill. var. Ambri kashmiri), and mushroom
(Agaricus bisporus). All the samples were mixed with Cys (100, 200 and 300 mg/kg.), AA(250,500 and
1000 mg/kg.), CA (250, 500 and 1000 mg/kg.) and SMB (100,200 and 300 mg/kg.) to assess their effect
on PPO. PPO activity was analyzed spectrophotometrically at 420 nm (30
0
C). The most effective PPO
inhibitors were AA and SMB and in combination with CA and Cys in all the samples tested. No
significant differences were observed for PPO activity among concentrations of Cys and CA when both
anti-browning agents were used alone or in combination and mixed with the samples.
Key words: Inhibition, Anti-browning agents, Banana, Apple and Mushroom Polyphenol oxidase,
Chemical treatment.
INTRODUCTION
Enzymatic browning is one of the most studied reactions in fruits and vegetables during
handling, processing and storage. The main enzyme involve in browning reaction is polyphenol
oxidase (PPO, Ec 1.14. 18.1). The activity of polyphenol oxidase has been extensively studied
and reported as the key factor in browning reaction
1
. PPO, also called tyrosinase, phenol oxidase,
cresolase are copper containing enzyme, synthesized in plants and stored in chloroplast
2
.
Polyphenol oxidase catalyzes the O-hydroxylation of monophenols to O- diphenols, which are
further catalyzed to produce O-quinones. Once formed, these quinones undergoes
Int. J. Chem. Sci.: 8(5), 2010
S551
polymerization reactions leading to the production of black, brown or red pigments
(Polyphenols) which is the cause of enzymatic browning in fruits and vegetables. So, it is
desirable to inactivate the PPO activity during preservation of foods as the enzymatic browning
change the physical appearance, sensory and nutritional quality of fruits and vegetables
3
. Now a
days, several methods have been used to inhibit the PPO activity, such as the use of Anti-
browning agents, removal of its one of the necessary component: O
2
, Cu
2+
etc or by thermal
processing to inactivate PPO activity is limited due to loss of sensory and nutritional quality of
fruits and vegetables
4
. Removal of oxygen from PPO can also check enzymatic browning but
browning may restart when oxygen is available
5
. So, the best way to prevent enzymatic
browning is the use of anti-browning agents. These agents act on the enzyme or react with the
substrate and/or products of enzymatic catalysis and thereby formation of browning pigment is
inhibited
6
. Several Anti-browning agents such as sulphites (Sulphur dioxide, sodium or
potassium metabisulfite, sodium or potassium bisulfite); acidulants such as citric, malic or
phosphoric acid; chelators (EDTA); reducing agents (ascorbic acid alone or in combination,
Cysteine); 4-hydroxyrecercinol etc have been extensively used for preventing browning of
foods
7,8
. Ascorbic acid reduces o-quinones to diphenols and prevents the formation of browning
pigments
9
. Sometimes ascorbic acid is oxidized completely and browning may occur due to
formation of melanins
10
. Acidifiers or acidulants can inhibit PPO activity by reducing pH and or
chelating copper in fruits and vegetables
11
. Cysteine reacts with O- quinones and forms a
colourless compound and thereby reduces PPO activity by delaying discolorations
12
. Another
anti-browning agent, 4- hydroxyresorcinol binds with PPO to form an inactive complex and
thereby reduces the browning reaction
13
. The objective of this study was to evaluate the anti-
browning (inhibition of PPO activity) effect of Cysteine, ascorbic acid, citric acid, sodium
metabisulfite alone or in combination, at three different pH (3.5, 4 and 4.5) in banana, apple and
mushroom.
EXPERIMENTAL
Materials and methods
Materials
Healthy, disease free Banana (Musa paradisiaca L. var. Kanthali), apple (Malus pumila
Mill. var. Ambri kashmiri), and mushroom (Agaricus bisporus) were purchased from the local
market.
Preparation of Crush
The materials were washed, held in ice water (1–2
0
C). Apple and banana were peeled,
de-seeded and cut into pieces. Mushrooms were sliced. Materials were blended in a domestic
blender to obtain a crush and kept in beakers (around 10–20 min) surrounded with ice water to
slow down discoloration. The crush was adjusted to desired pH (3.5, 4.0, or 4.5) with phosphoric
acid immediately after preparation. Crush (50 g) was weighed and mixed with sodium
metabisulphite (E. Merck), L-ascorbic acid (E. Merck), or L-cysteine hydrochloride hydrate (E.
Merck) and Citric acid (E. Merck) alone, or in combination as presented in Table 1. Each crush
A. Samanta et al.: Inhibition of Polyphenol Oxidase….
S552
was weighed (35 g) and placed into a sterile sample bag with remaining air evacuated and tabs
folded 8 times to seal the opening. Bags were then held in ice water until analysis for PPO
activity. The study was made in duplicate.
Table 1: Combination of different anti-browning agents used in this experiment
Treatments - Anti-browning agents
Cys (mg/kg) AA (mg/kg) CA(mg/kg)
SMB
(mg/kg)
0 100 200 300 250 500 1000 250 500 1000
0
0 4 5 6 16 17 18 28 29 30
100
1 7 8 9 19 20 21 31 32 33
200
2 10 11 12 22 23 24 34 35 36
300
3 13 14 15 25 26 27 37 35 36
PPO assay
PPO activity was assessed spectrophotometrically at 420 nm (30
0
C) in a UV-Vis
spectrophotometer (Elico, India)
14
. The PPO extract was obtained by mixing 10 g of apple,
banana and mushroom crush with 10 mL of sodium phosphate buffer (pH 6.6) at 5
o
C and
centrifuging at 4000 rpm for 40 min at 4
o
C. The extract was filtered with Whatman paper No. 1.
The supernatant was denominated as enzyme extract. The reaction mixture consisted of 2.2 mL
of buffer, 0.5 mL of catechol (0.175 mol/L), and 0.3 mL of enzyme extract. After mixing the
mixture the absorbance was recorded every 15 s for 3 min. The linear portion obtained in
plotting absorbance as a function of time was used to compute PPO activity. One unit of PPO
activity was defined as 0.001 ∆A420/min/mL. All extracts were analysed in triplicate.
Statistical analysis
Data analysis was carried out using a Microsoft Excel 2003 and analysis of variance
(ANOVA) was performed with the SPSS 12.0 software. *P < 0.05 was selected as the decision
for significant differences.
RESULTS AND DISCUSSION
Banana, apple and mushroom crush containing SMB, Cys, AA and CA
PPO activity
PPO activity in banana, apple and mushroom crush at different pH (3.5,4.0 and 4.5) and
different concentrations of anti-browning agents were presented in Table 2,3 and 4 respectively.
From the tables, it can be concluded that the PPO activity decreased as the pH of the crush
materials decreased in case of all the three species studied. It was also observed that the PPO
activity was drastically decreased at pH 4 and pH 3.5 in all the three species, as compared to pH
4.5 (Fig. 1, 2 and 3). As the PPO activity is maximally observed between pH 5 and 7
13
, low pH
Int. J. Chem. Sci.: 8(5), 2010
S553
may inhibit, or minimize PPO activity due to acidification
11
. According to McEvily et al.
9
, PPO
was completely inhibited at a pH below 3. But fruit products generally do not possess this much
low pH and in this low pH the taste of such products become changed and it may be extremely
acidic for regular consumption. The anti-browning effect of SMB, AA, CA and Cys (used alone)
in banana, apple and mushroom crush were presented in Table 2, 3 and 4 respectively and a
comparative study was also carried out to determine the potentiality of different selected anti-
browning agents and were presented in Fig. 1 (for Banana), Fig. 2 (for Apple) and Fig. 3 (for
Mushroom). In general, maximum inhibition activity of PPO were observed at a concentration of
200 mg/kg SMB at pH-3.5 and 1000 mg/kg AA at pH-3.5 in case of banana crush; 300 mg/kg
SMB at pH-3.5 and 1000 mg/kg AA at pH-3.5 in case of apple crush; 200 mg/kg SMB at pH-4
and 1000 mg/kg AA at pH- 4 and 300 mg/kg cysteine at pH-4 in case of mushroom crush.
McEvily et al.
9
suggested that ascorbic acid inhibits PPO activity by reducing o-quinones to
diphenols and cysteine reacts with o-quinones to form a colourless compound and thereby
reduces PPO activity. AA contributes both weak acidic and strong reducing properties
15
that may
reduce the pH of crush. However, from the two-way ANOVA study, we can conclude that the
residual PPO activity at different pH and at different concentration of selected anti-browning
agents were significantly different from control (*P < 0.05).
Table 2: Residual PPO activity in banana crush containing different anti-browning agents
at various pH
Enzyme activity units
Anti-browning agents
PH
Species Concentration (mg/Kg) 3.5 4.0 4.5
Control
0.000
14.5±1.2 32.1±1.1 248.4±2.4
100 8.7±1.8 10.4±1.2 102.6±1.9
200 4.0±1.3 5.6±1.4 81.3±1.3
Sodium
metabisulfite
300 10.6±1.6 21.7±1.5 138.5±1.4
250 8.2±1.1 6.5±1.5 90.6±1.4
500 5.4±1.1 7.6±2.1 78.4±1.4
Ascorbic acid
1000 4.2±1.2 3.8±1.8 40.6±1.2
250 9.4±1.4 11.8±2.0 122.8±1.3
500 6.2±1.4 8.6±1.1 106.2±1.9 Citric acid
1000 5.2±1.6 5.8±1.0 92.8±1.4
100 8.6±1.9 10.8±1.8 118.2±1.2
200 6.0±1.1 5.4±1.2 84.6±1.8
Cystein
300 4.8±1.6 3.8±1.8 68.2±1.5
A. Samanta et al.: Inhibition of Polyphenol Oxidase….
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Table 3: Residual PPO activity in apple crush containing different anti-browning agents at
various pH
Enzyme activity Units
Anti-browning agents
PH
Species Concentration (mg/Kg) 3.5 4.0 4.5
Control
0.000 11.61±1.1 25.68±2.1 244.41±1.5
100 6.99±1.1 8.22±2.6 95.53±1.6
200 3.22±1.6 18.76±1.6 79.93±1.2
Sodium
metabisulfite
300 8.48±1.5 4.96±1.7 72.53±1.6
250 6.66±1.9 5.05±1.6 83.34±1.7
500 4.32±1.8 6.66±1.7 75.53±1.4
Ascorbic acid
1000 2.68±1.4 4.42±1.1 34.4±61.2
250 7.53±1.5 9.08±1.6 112.16±1.4
500 4.66±1.5 7.06±1.9 102.23±2.2
Citric acid
1000 4.16±1.6 3.83±1.7 87.77±2.6
100 8.44±1.3 10.22±1.5 108.33±1.5
200 7.03±1.5 8.71 ±1.6 77.56±1.9
Cysteine
300 6.22±1.3 7.43±1.8 62.19±1.4
Table 4: Residual PPO activity in mushroom crush containing different anti-browning
agents at various pH
Enzyme activity Units
Anti-browning agents
PH
Species Concentration (mg/Kg) 3.5 4.0 4.5
Control
0.000 39.66±1.1 41.77±2.3 322.22±1.4
100 98.33±1.3 17.87±1.9 174.29±1.9
200 20.5±1.3 7.78±1.1 105.56±1.5
Sodium
metabisulfite
300 13.60±1.9 36.56±1.7 98.02±1.4
250 10.03±1.5 10.98±1.2 117.77±1.3
500 36.66±1.8 12.84±1.8 132.49±1.8
Ascorbic acid
1000 22.99±1.9 6.35±1.4 68.61±1.6
250 11.96±1.6 19.26±1.2 184.22±1.9
500 13.62±1.4 11.78±1.3 137.8±1.1
Citric acid
1000 6.23±1.6 12.25±1.1 120.66±1.6
Cont…
Int. J. Chem. Sci.: 8(5), 2010
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Enzyme activity Units
Anti-browning agents
PH
Species Concentration (mg/Kg) 3.5 4.0 4.5
100 95.33±1.6 31.75±1.1 154.05±1.6
200 46.29±1.1 7.06±1.4 110.12±1.4
Cysteine
300 29.77±1.6 5.02±1.5 88.66±2.8
0
50
100
150
200
250
300
CP1P2P3Q1Q2Q3R1R2R3S1S2S3
Different antibrowning agent at different concentration
Enzy
m
e
Activity Units
pH 3.5
pH 4
pH 4.5
Fig.1: Residual PPO activity in banana treated with different anti-browning agents
C = Control; P1, P2 and P3 = SMB (100, 200 and 300 mg/kg ); Q1,Q2 and Q3 = AA (100,200 and 300
mg/kg ); R1, R2 and R3 = CA (100, 200 and 300 mg/kg ); S1,S2 and S3 = Cys (100,200 and 300 mg/kg )
0
50
100
150
200
250
300
C P1P2P3Q1Q2Q3R1R2R3S1S2S3
Different antibrownin
g
a
g
ent at different concentration
Enzy
m
e
activity units
pH 3.5
pH 4
pH 4.5
Fig. 2: Residual PPO activity in apple treated with different anti-browning agents. C = Control;
P1, P2 and P3 = SMB (100, 200 and 300 mg/kg ); Q1,Q2 and Q3 = AA(100, 200 and 300 mg/kg );
R1,R2 and R3 = CA(100 ,200 and 300 mg/kg ); S1,S2 and S3 = Cys (100 ,200 and 300 mg/kg )
A. Samanta et al.: Inhibition of Polyphenol Oxidase….
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0
50
100
150
200
250
300
350
C P1P2P3Q1Q2Q3R1R2R3S1S2S3
Different antibrowning agent at different concentration
Enzy
m
e
activity units
pH 3.5
pH 4
pH 4.5
Fig. 3: Residual PPO activity in mushroom treated with different anti-browning agents
C = Control; P1,P2 and P3 = SMB(100, 200 and 300 mg/kg ); Q1,Q2 and Q3 = AA(100, 200 and 300
mg/kg ); R1,R2 and R3 = CA(100, 200 and 300 mg/kg ); S1,S2 and S3 = Cys (100, 200 and 300 mg/kg ).
Banana, apple and mushroom with SMB/Cys, SMB/AA and SMB/CA
PPO activity
Table 5, 6 and 7 represents the residual PPO activity in banana, apple and mushroom
crush respectively (pH 3.5) containing different combinations of anti-browning agents. The
lowest PPO activity in banana crush was obtained after adding combinations of SMB/Cys
(200/300 mg/kg) and SMB/AA (100/500 mg/kg) (Table 5). The combinations of SMB/Cys
(100/200 mg/kg) and SMB/AA (100/250 mg/kg) were most suitable in reducing PPO activity in
apple (Table 6). From Table 7, it was observed that SMB/AA (100/500 mg/kg) and SMB/CA
(100/250 mg/kg) combinations in mushroom crush showed reduced PPO activity.
Table 5: Residual PPO activity in banana crush containing different anti-browning agents
at various pH
Enzyme activity units
Cys (mg/Kg) AA (mg/Kg) CA (mg/Kg)
SMB
100 200 300 250 500 1000 250 500 1000
100
4.6±1.2 4.8±1.3 8.1±1.1 3.8±1.7 3.2±1.3 5.4±1.7 5.2±1.1 6.2±1.2 8.9±1.6
200
5.5±1.2 6.0±2.1 4.1±1.4 5.1±1.6 8.4±2.2 8.8±1.7 7.6±1.9 5.6±1.7 9.2±1.1
300
8.9±1.7 11.6±1.1 7.6±1.4 8.8±1.8 9.6±1.7 9.4±1.8 11.8±1.2 10.6±1.4 11.8±1.5
Int. J. Chem. Sci.: 8(5), 2010
S557
Table: 6. Residual PPO activity in apple crush containing different anti-browning agents at
various pH
Enzyme activity units
Cys (mg/Kg) AA (mg/Kg) CA (mg/Kg)
SMB
100 200 300 250 500 1000 250 500 1000
100
3.6±1.9 3.4±1.2 6.5±1.6 3.2±1.3 3.9±1.6 4.4±1.1 4.1±1.7 4.9±1.4 6.9±1.4
200
4.5±2.1 5.1±1.2 3.3±1.5 4.1±2.2 6.7±1.7 7.2±2.1 6.0±1.5 4.9±1.8 7.2±1.8
300
7.1±1.1 9.1±1.3 5.9±2.1 7.0±2.5 7.4±1.4 7.8±1.8 9.4±1.1 8.4±2.1 9.4±1.1
Table 7: Residual PPO activity in mushroom crush containing different anti-browning
agents at various pH
Enzyme activity units
Cys (mg/Kg) AA (mg/Kg) CA (mg/Kg)
SMB
100 200 300 250 500 1000 250 500 1000
100
5.8±1.3 6.4±2.3 10.7±1.4 4.2±1.7 3.7±1.1 6.8±1.7 3.3±1.2 5.5±1.6 11.2±1.4
200
7.1±1.7 7.6±1.6 5.3±1.1 6.1±1.5 11.2±1.7 11.9±1.4 6.7±1.8 5.9±1.1 9.1±1.2
300
11.5±2.3 14.0±2.1 10.2±1.1 11.6±1.7 12.4±1.5 12.2±1.1 11.6±1.3 8.2±1.3 12.1±1.7
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