ISSN: 2410-8790
Olusola and Oluwatosin / Current Science Perspectives 2(2) (2016) 14-18
Current Science Perspectives 2(2) (2016) 14-18
iscientic.org.
Extraction of polyphenol oxidase from green and red apple fruits and the effect of pH
variation on the activity of the enzyme
Ladokun Olusola and Arojojoye Oluwatosin*
Lead City University, Department of Biochemistry, P.O. Box 30678, Secretariat, Ibadan, Oyo State, Nigeria
*Corresponding author’s E-mail: [email protected]
A R T I C L E
I N F O
Article type:
Research article
Article history:
Received October 2015
Accepted November 2015
April 2016 Issue
Keywords:
Polyphenol oxidase
Red apple
Green apple
Optimum PH
A
B
S T
R
A
C
T
Polyphenol oxidase is present in the majority of plant tissues and it is involved in
adverse browning of plant products. Crude polyphenol oxidase (PPO) was extracted
from flesh and peels of green and red apple fruits and the concentration of the enzyme
in the flesh and peels of the fruits was determined spectrophotometrically. The activity
of the enzyme in the green apple peel, green apple flesh, red apple peel and red apple
flesh were found to be 2.15, 1.30, 0.70 and 2.65 µmolmin-1cm-3 respectively. The
effect of pH on the stability of the enzyme’s activity was also investigated. Catechol at
a concentration of 20 mM was used as a substrate while sodium acetate buffer (0.2M)
with a pH range of 3.6-5.6 and sodium phosphate buffer (0.2M) with a pH range of
6.0-8.0 were used to monitor the effect of pH on the activity of the enzyme. From the
results obtained, optimum pH for crude PPO extracted from the green apple peel,
green apple flesh, red apple peel and red apple flesh were 7.6, 6.4, 8.4 and 8.0
respectively. Increase or decrease of pH from this range caused decrease in the activity
of the enzyme. This can therefore be a good way of controlling undesirable changes in
these fruits.
© 2016 International Scientific Organization: All rights reserved.
Capsule Summary: The effect of pH on enzyme activity from apple and it was found that by adjusting pH, the red and green apples
can be used in controlling undesirable changes in the fruits.
Cite This Article As: Ladokun Olusola and Arojojoye Oluwatosin. 2016. Extraction of polyphenol oxidase from green and red apple
fruits and the effect of pH variation on the activity of the enzyme. Current Science Perspectives 2(2) 14-18
reactive oxygen molecules bound to a carbon atom by two
double bonds. Polyphenol oxidase catalyzes two reactions; the
first, a hydroxylation of monophenols to diphenols, which is
relatively slow and results in colourless products. The second,
the oxidation of diphenols to quinines which is rapid and gives
coloured products (Queiroz et al., 2008).
Polyphenol oxidase (PPO) causes oxidative browning in
many food products (Chi et al., 2014).Enzymatic browning is a
significant problem in a number of fruits and vegetables resulting
in discoloration of fruits and vegetables. This occurs as a result
of conversion of phenolic compounds to o-quinones which
subsequently polymerize to be a brown or dark pigment.
Polyphenol oxidase has received much attention from researchers
in the field of plant physiology and food science because of its
INTRODUCTION
Polyphenol oxidase also known as tyrosinase, polyphenolase,
phenolase, catechol oxidase, cresolase, or catecholase which is
widely found in nature is typically present in the majority of
plant tissues (Whitaker 1994, 1996; Fraignier et al., 1995; Haruta
et al., 1999). Polyphenol oxidases are a widespread group of
enzymes found in plants, fungi, bacteria, and animals. In plants,
these enzymes are usually found in the chloroplasts, although
they can be released from this compartment during ripening or
senescence.
The enzymes contain copper at their active site. This metal
ion enables them to oxidize the phenolic group of an aromatic
compound to a reaction group known as a quinone. Quinones are
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Olusola and Oluwatosin / Current Science Perspectives 2(2) (2016) 14-18
involvement in adverse browning of plant products (Ruhiye and
Maurice, 2003).
Plants and fruits account for a substantial fraction of the
world's agricultural output (Ali et al., 2015; Ashraf et al., 2015;
Asif, 2015a, b, c, d, e, f, g, h, i, 2016; Hussain et al., 2016; John
et al., 2015; Mensah and Golomeke, 2015) and some (such as the
apple and the pomegranate) have acquired extensive cultural and
symbolic meanings. Wounds inflicted during the preparation of
fresh-cut on fruits and tubers promote many physical and
physiological changes that hasten loss of product quality.
Foremost among these, are the removal of the protective
epidermal layer and/or exposure of internal cells. These changes
not only facilitate water loss, but also provide an easy entry for
microbial pathogens and chemical contaminants. When fruits or
tubers are peeled or cut, enzymes contained in the plant cells are
released. One of these enzymes is polyphenoloxidase which is
involved in enzymatic browning which occurs readily at warm
temperatures when the pH is between 5.0 and 7.0 (Cisneros,
1995).
Enzymatic browning affects nutritional properties, flavor
and texture of foods and feeds during storage or processing and
is therefore detrimental to food quality. Browning and
discoloration causes substantial losses in a wide range of fresh
and processed fruits and tubers. Traditionally, browning in foods
has been controlled by using sulfating agents; such food
additives have been used in a wide range of fresh, frozen and
processed food products.
In this study, polyphenoloxidase was extracted from green
and red apple fruits and the concentration of the enzyme was
determined in the flesh and peels of these fruits, the optimal pH
of the enzyme was also determined to monitor the effect of PH
on the activity of the enzyme.
20 C. Polyphenol oxidase activity was determined according to
the method of Ying and Zhang, 2008 by measuring the increase
in absorbance at 420nm with spectrophotometer. The sample
cuvette contained 2.0cm3 of catechol, 0.9cm3 of 0.2M sodium
acetate buffer pH 4.0 and 0.1cm3 of enzyme solution. Reference
cuvette (blank) contained 2.0cm3 of the same substrate solution
and 1.0cm3 of 0.2M sodium acetate buffer. Each sample was
assayed in triplicates.
Effect of pH on polyphenol oxidase activity and stability
0.2M sodium acetate buffer of pH range of 3.6 -5.6 and 0.2M
sodium phosphate buffer of pH range 6.0-8.0 and catechol
(20mM) were used to determine the effect of pH on PPO activity
and stability, 0.1cm3 of enzyme solution was incubated in 0.9cm3
of the buffer solutions for 10 hours at 40C. The enzyme activity
was measured according to the method described by Ying and
Zhang, (2008).
RESULTS AND DISCUSSION
Polyphenol oxidas activities in red and green apple fruits are
shown in Table 1, effect of pH on the activity of crude PPO
extracted from green apple peels is depicted in figure 1, effect of
pH on the activity of crude PPO extracted from green apple flesh
is depicted in figure 2, effect of pH on the activity of crude PPO
extracted from red apple peels is depicted in figure 3 and the
effect of pH on the activity of crude PPO extracted from red
apple flesh is depicted in figure 1.
Polyphenol oxidase (PPO) catalyzes the oxidation of
phenolics to quinones. The subsequent non-enzymatic
polymerization of the quinones leads to formation of brown
pigments that are the cause of post-harvest deterioration and loss
of quality in many economically important crops (Thygesen and
Robinson, 1995).
Polyphenol oxidase (PPO) -catalyzed browning reactions are
of significant importance in the fruit and vegetable industry
(Yoruk and Marshall, 2003). Losses caused by the browning
resulting from PPO catalyzed-oxidations probably account for
50% of the losses of industrial production of fruits and
vegetables (Holderbaum et al., 2010). The dark colour coupled
with the off-tastes in juices and vegetables and the resulting
changes in texture of fruits and vegetables that undergo browning
discourage consumer from purchasing them.
Some commercially important edible plant products
susceptible to adverse browning reactions include fruits such as
apple (Yemenicioglu et al., 1997), avocado (Espin et al., 1997a),
banana (Gooding et al., 2001), cucumber (Miller et al., 1990),
grape (Rathjen and Robinson, 1992), pineapple (Das et al.,
1997). However, browning is beneficial to some extent in some
instances as it enhances the quality of beverages through its
forming flavorful products, for example in the processing of
black tea (Ullah, 1991), coffee (Amorim and Melo,1991) and
cocoa (Lopez and Dimick, 1991).
Since PPO is involved in enzymatic browning, this
phenomenon can be prevented by inhibiting the enzyme’s
activity. Owing to its tremendous economic impact to the food
industry, inhibition of PPO in some food products has been
studied (Kim et al., 2000). Concentration is one of the factors
that affect enzyme’s activity. Much work has been done on the
determination of PPO activity in some selected fruits with a
paucity of report on their peels. Therefore in this study, the
MATERIAL AND METHODS
Source of fruits
Green and red apple fruits were purchased from Oje market in
Ibadan, Oyo State, Nigeria.
Reagents
0.1M phosphate buffer, Ascorbic acid, polyvinylpolypyrrolidone
(PVPP), Triton 100 (Tx -100), 20mM catechol (pyrocatechol),
0.2M Sodium acetate buffer, 0.2M Sodium phosphate
Extraction and determination of polyphenol oxidase activity
The extraction was carried out at 40C in order to prevent loss of
enzyme activity. Polyphenol oxidase was extracted from the
fruits using Triton x 100 detergent. This detergent has been used
for the extraction and solubilisation of polyphenol oxidase in
several fruits. Ascorbic acid was used as reducing agent of
endogenous phenolic compounds found in the fruits. Finally,
polyvinylpolypyrrolidone (PVPP) was used to prevent quinone
formation, as it reacts with the proteins that are present in the
enzyme. 150g of samples (peels and flesh) was blended with
300ml 0.1M phosphate buffer, 0.2g of abscorbic acid, 3% PVPP
and Tx-100 for 10minutes.The sample was centrifuged at
18,000rpm for 20 min at 4 0C with cold centrifuge and the
supernatant was collected, filtered with whatman No. 4 paper and
the filtrate was taken as the crude enzyme extract and stored at www.bosaljournals/csp/
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ISSN: 2410-8790
Olusola and Oluwatosin / Current Science Perspectives 2(2) (2016) 14-18
G r e e n a p p le p e e ls
R e d a p p le p e e ls
25
30
cm
-3
-3
-1
20
A c tiv it y (  m o lm in
cm
-1
A c tiv it y (  m o lm in
iscientic.org.
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10
5
0
0
2
4
6
8
20
10
0
10
0
pH
2
4
6
8
10
pH
Fig. 3: Activity of crude PPO extracted from red apple peels (Malus
domestica) against pH
Fig. 1: Activity of crude PPO extracted from green apple peels
(Malus domestica) against pH
G r e e n a p p le f l e s h
R e d a p p le f le s h
60
-1
A c tiv it y (  m o lm in
-1
A c tiv ity ( m o lm in c m
cm
-3
-3
30
40
20
0
20
10
0
0
2
4
6
8
0
10
2
4
6
8
10
pH
pH
Fig. 2: Activity of crude PPO extracted from green apple flesh
(Malus domestica) against pH
Fig. 4: Activity of crude PPO extracted from red apple flesh (Malus
domestica) against pH
concentration of PPO in both the flesh and peels of red and green
apples was determined and this is proportional to the enzyme’s
activity in the flesh and the peels. The concentration of the
enzyme in the flesh compared to the peels of the apples revealed
that the enzyme’s activity was lower in the green apple flesh than
the peel but higher in red apple flesh than in the peel, this result
was confirmed by the rate of browning reaction observed during
peeling. The red apple flesh had the highest PPO activity of 2.65;
this high PPO activity could be responsible for the rapid change
in colouration observed in the red apple flesh during peeling. The
activity of the enzyme in the green apple peel was 2.15; the
activity in the green apple flesh was 1.3 while the enzyme’s
activity in the red apple peel was 0.75.
PH is another factor which affects the rate of an enzyme
catalyzed reaction. The changes in ionization of prototropic
groups in the active site of an enzyme at lower acid and higher
alkali pH values may prevent proper conformation of the active
site, binding of substrates, and/or catalysis of the reaction
(Whitaker 1994). An optimum pH is the pH at which an enzyme
shows its maximum activity. Therefore for any enzyme, when
the [H+] concentration of the reaction medium is increased above
or decreased below the optimum pH, the activity tends to
decrease. The optimum pH for an enzyme depends on the
substrate in the activity assay (Arnok 2010). From the result
obtained in this study, the optimum pH for crude PPO extracted
from the green apple peel, green apple flesh, red apple peel and
red apple flesh were 7.6, 6.4, 8.4 and 8.0 respectively as depicted
in figures 4.1-4.4 above. Optimum PH for some plants has also
been reported. Heimdal et al., 1994 reported optimum PH of 5.08.0 for PPO activity in lettuce. Sheptovitsky and Brudvig, 1996
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Asif, M., 2015f. The impact of dietary fat and polyunsaturated
fatty acids on chronic renal diseases. Current Science
Perspectives 1, 51-61.
Asif, M., 2015g. Pharmacological activities and phytochemistry
of various plant containing coumarin derivatives. Current
Science 1, 77-90.
Asif, M., 2015h. Pharmacologically potentials of different
substituted coumarin derivatives. Chemistry International 1,
1-11.
Asif, M., 2015i. Role of some nutritional complements and
biological supplements in the management of epilepsy.
Current Science Perspectives 1, 1-1.
Asif, M., 2016. A review on recent advances and potential
pharmacological activities of versatile chalchone molecule.
Chemistry International 2, 1-18.
Barbaros, D., Ahmet, C., Nese, A., Asim, K., Saadettin, G.2002.
Characterization of polphenol oxidase from medlar fruits
(Mespilus germanica L. Rosaceae). Food Chem. 77, 1-7.
Chi, M., Bhagwat, B., Lane, W. D., Tang, G., Su, Y., Sun, R.,
Oomah, B. D., Wiersma, P. A., Xiang, Y., 2014. Reduced
polyphenol oxidase gene expression and enzymatic
browning in potato (Solanum tuberosum L.) with artificial
microRNAs. BMC Plant Biology 14, 62.
Cisneros-Zevallos, L., Saltveit, M.E. and Krochta, J.M., 1995.
Mechanism of surface white discoloration of peeled
(minimally processed) carrots during storage. J. Food Sci.
60, 320-323, 333.
Espin, J.C., Trujano, M.F., Tudela, J., Garcia-Canovas, F.,
1997a. Monophenolase activity of polyphenol oxidase from
Haas avocado. J. Agric. Food Chem. 45, 1091-1096.
Fraignier, M., Marques, L., Fleuriet, A. and Macheix, J., 1995.
Biochemical and immunochemical characteristics of
polyphenol oxidase from different fruits of Prunus. J. Agric.
Food Chem. 43, 2375-2380.
Gawlik-Dziki, U., Złotek, U., Świeca, M.,
2007.
Characterization of polyphenol oxidase from butter lettuce
(Luctuca sativa var. capitata L.). Food Chemistry 107, 129135.
Gooding, P.S., Bird, C. and Robinson, S.P. 2001. Molecular
cloning and characterization of banana fruit polyphenol
oxidase. Planta 213, 748-757.
Haruta, M., Murata, M., Kadokura, H., Homma, S., 1999.
Immunological and molecular comparison of polyphenol
oxidase in Rosaceae fruit trees. Phytochemistry 50, 10211025.
Heimdal, H., Larsen, L.M., Poll, L., 1994. Characterization of
polyphenol oxidase from photosynthetic and vascular lettuce
tissues (Lactucu saliva). J. Agric. Food. Chem. 42, 14281433.
Holderbaum, D.F., Kon, T., Kudo, T., Guerra, M.P., 2010.
Enzymatic browning, polyphenol oxidase activity, and
polyphenols in four apple cultivars, dynamics during fruit
development. Hort Sci. 45(8), 1150-1154.
Hussain, F., Shahid, M., Javed, K., 2016. Antioxidant,
antiglycation and alpha Amylase inhibitory activities of
Cassia absus seeds. Current Science Perspectives 2, 5-9.
Jiang, Y., 1999. Purification and some properties of polyphenol
oxidase of longan fruit. Food Chemistry 66, 75-79.
John, K.M., Kwoseh, C., Banahene, N., Atuilik, S.A., Oppong,
D., Borigu, M., 2015. Assessment of the Antimicrobial
Activities of the Secondary Metabolites Produced by Pure
Table 1: Polyphenol oxidas activity in red and green apple
fruits
Samples
PPO Activities
(µmol min-1cm-3)
Green apple peels
2.15
Green apple flesh
1.30
Red apple peels
0.75
Red apple flesh
2.65
reported PH optimum of around 8.0 for PPO activity in spinach.
Gawlik-Dziki et al., 2007 reported an optimum pH value 0f 5.5
for PPO activity in butter lettuce using catechol as substrate.
Jiang, 1999 also reported an optimum pH value 0f 6.5 for PPO
activity in longan fruit using 4-methycatechol as substrate and
Barbaros et al., 2002 reported an optimum pH value of 6.5 for
PPO activity in medlar.
CONCLUSIONS
In present investigation, crude polyphenol oxidase was extracted
from flesh and peels of green and red apple fruits and the
concentration of the enzyme in the flesh and peels of the fruits
was determined. Based on results, it was concluded that the
effect of pH on the stability of the enzyme’s activity was
considerable. The pH values beyond the optimum value affected
the enzyme activity and therefore, the adjustment of pH can
prevent undesirable browning in the fruits. This will help to
increase the shelf life of fruits and prevent unnecessary wastage.
REFERENCES
Ali, A., Khalil-ur-Rahman, Jamil, A., Jahan, N., Tahir, A., 2015.
Phyto-constituents, DNA protection and cytotoxic potential
of Rheum emodi. Current Science Perspectives 1, 102-106.
Amorim, H.W.D., Silva, D.M., 1968. Relationship between the
polyphenol oxidase activity of coffee beans and the quality
of the beverage. Nature 219, 381-382.
Ashraf, M.W., Muhammad, B., Munawar, I., 2015. Antiglycation
activity of vegetables aqueous and methanolic extracts.
Current Science Perspectives 1, 12-15.
Asif, M., 2015a. Anti-neuropathic and anticonvulsant activities
of various substituted triazoles analogues. Chemistry
International 1, 174-183.
Asif, M., 2015b. Anti-tubercular activity of some six membered
heterocycle compounds. Chemistry International 1, 134-163.
Asif, M., 2015c. Antiviral and antiparasitic activities of various
substituted triazole derivatives: A mini. Chemistry
International 1, 71-80.
Asif, M., 2015d. Chemical characteristics and nutritional
potentials of unsaturated fatty acids. Chemistry International
1, 118-133.
Asif, M., 2015e. Chemistry and antioxidant activity of plants
containing some phenolic compounds. Chemistry
International 1, 35-52.
www.bosaljournals/csp/
iscientic.org.
17
[email protected]
ISSN: 2410-8790
Olusola and Oluwatosin / Current Science Perspectives 2(2) (2016) 14-18
iscientic.org.
Cultured Trichoderma koningii, Rhizopus stolonifer and
Fusarium oxysporum. Current Science Perspectives 1, 96101.
Kim, J., Marshall, M.R., Wei, C., 2000. Polyphenoloxidase. In
Seafood Enzymes Utilization and Influence on Postharvest
Seafood Qualiq, (N.F. Haard and B.K. Simpson, eds.), p
271-315,
Lopez, A.S., Dimick, P.S., 1991. Enzymes involved in cocoa
curing. In Food Enzymology. (P.F. Fox, ed.), p 211-236,
Elsevier Science Publishing, New York.
Marcel Dekker, NewYork.
Mensah, J.K., Golomeke, D., 2015. Antioxidant and
antimicrobial activities of the extracts of the Calyx of
Hibiscus Sabdariffa Linn. Current Science Perspectives 1,
69-76.
Miller, A.R., Kelley, T.J., Mujer, C.V., 1990. Anodic peroxidase
isoenzymes and polyphenol oxidase activity from cucumber
fruit, tissue and substrate specificity. Phytochemistry 29,
705-709.
Queiroz, C., Lopes, M. L. M., Fialho, E., Valente-Mesquita, V.
L., 2008. Polyphenol oxidase, Characteristics and
mechanisms of browning control. Food Reviews
International 24(4), 361-375.
Rathjen, A.H., Robinson, S.P., 1992. Aberrant processing of
polyphenol oxidase in a variegated grapevine mutant. Plant
Physiology 99, 1619-1625.
Sheptovitsky, Y.G., Brudvig, G.W., 1996. Isolation and
characterization of spinach photosystem I1 membraneassociated catalase and polyphenol oxidase. Biochemistry
35, 16255- 16263.
Thygesen, P.W., Dry, I.B., Robinson, S.P., 1995. Polyphenol
oxidase in potato. A multigene family that exhibits
differential expression patterns. Plant Physiol. 109 (2), 525531.
Ullah, M.R., 1991. Tea. In Food Enzymotogy, (P.F. Fox, ed.).p
163-187, Elsevier Science Publishing, New York.
Whitaker, J.R., 1994. Principles of Enzymology for the Food
Sciences, Second Ed., p 271-556, Marcel Dekker, New
York.
Whitaker, J.R., 1996. Polyphenol oxidase. In Food Chemistry,
(O.R. Fennema, ed.), p 492-494, Marcel Dekker, New York.
Yemenicioglu, A., Ozkan, M. and Cemeroglu, B., 1997. Heat
inactivation kinetics of apple polyphenoloxidase and of its
latent form. Journal of Food Science 62, 508-510.
Ying, Y., Zhang, W., 2008. Some properties of polyphenol
oxidase from lily. International Journal of Food Science and
Technology 43, 102–107.
Yoruk, R., Marshall, M. R., 2003. Physicochemical properties
and function of plant polyphenol oxidase, A REVIEW.
Journal of Food Biochemistry 27(5), 361-422.
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