Food Chemistry 125 (2011) 128–132

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Polyphenol oxidase activity, phenolic acid composition and browning

in cashew apple (Anacardium occidentale, L.) after processing
Christiane Queiroz a, Antonio Jorge Ribeiro da Silva b, Maria Lúcia Mendes Lopes a,
Eliane Fialho a, Vera Lúcia Valente-Mesquita a,⇑
a
Departamento de Nutrição Básica e Experimental, Instituto de Nutrição Josué de Castro, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 393 RJ 21941-590, Brazil
b
Núcleo de Pesquisa em Produtos Naturais (NPPN) Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 393 RJ 21941-590, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: This study describes the extraction and characterisation of cashew apple polyphenol oxidase (PPO) and
Received 26 February 2010 the effect of wounding on cashew apple phenolic acid composition, PPO activity and fruit browning. Puri-
Received in revised form 23 June 2010 fication factor was 59 at 95% (NH4)2SO4 saturation. For PPO activity, the optimal substrate was catechol
Accepted 23 August 2010
and the optimum pH was 6.5. PPO Km and Vmax values were 18.8 mM and 13.6 U min 1 ml 1, respectively.
Ascorbic acid, citric acid, sodium sulphite and sodium metabisulphite decreased PPO activity, while
sodium chloride increased PPO activity. Wounding at 2 °C and 27 °C for 24 h increased PPO activity
Keywords:
but storage at 40 °C reduced PPO activity. Gallic acid, protocatechuic acid and cinnamic acid (free and
Cashew apple
Polyphenol oxidase
conjugate) were identified in cashew apple juice. Cutting and subsequent storage at 40 °C hydrolysed cin-
Injury namic acid. 5-Hydroxymethylfurfural content in cashew apple juice increased after injury and storage at
Phenolic acids higher temperatures, indicating non-enzymatic browning.
Browning Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The cashew tree (Anacardium occidentale, L.) is native to Brazil

Polyphenol oxidase (PPO EC. 1.14.18.1 or EC 1.10.3.2) consists of
a group of copper-containing enzymes that are widely distributed
among plant species and are responsible for browning in plant tis-
sues. PPO catalyses the oxidation of polyphenols to quinones that
react non-enzymatically to produce coloured pigments. For most
plant tissues, PPO is compartmentalised in plastids, whereas phe-
nolic substrates are located in the vacuoles. PPO activation occurs
only when these compartments are disrupted after tissue wound-
ing (Mayer, 1987; Yoruk & Marshall, 2006). PPO exists in a latent
state and can be activated by treatment with several agents,
including sodium dodecyl sulphate (SDS) (Sellés-Marchart, Casa-
do-Vela, & Bru-Martínez, 2006).
Browning reactions change sensory properties, reduce nutri-
tional quality and decrease consumer acceptance. PPO has been
purified from different sources, including Barbados cherry (Kumar,
Mohan, & Murugan, 2008), banana (Ünal, 2007), grape (Rapeanu,
Loey, Smout, & Hendrickx, 2006) and other plants (Queiroz, Lopes,
Fialho, & Valente-Mesquita, 2008; Yoruk & Marshall, 2006), but has
not been previously characterised in cashew apple.
⇑ Corresponding author. Tel.: +55 (21) 25626449.
E-mail address: valentem@nutricao.ufrj.br (V.L. Valente-Mesquita).
and its culture has a large socioeconomic importance for the
northeast region of the country. The cashew nut is defined
botanically as a fruit and is the main exported product of the
cashew tree (IBGE, 2009). Peduncle, also called cashew apple,
has a high content of ascorbic acid (Lavinas, Almeida, Miguel,
Lopes, & Valente-Mesquita, 2006) and carotenoids and displays
antioxidant capacity (Barreto, Souza, Azeredo, & Mercadante,
2007). Phenolic compounds, such as phenolic acids, flavonoids
and tannins, were also identified in cashew apple (Brito, Araújo,
Lin, & Harnly, 2007; Michoudjehoun-Mestres et al., 2009). How-
ever, despite its nutritional composition, cashew apple does not
have high commercial importance due its astringency and high
perishability. Oxidation of phenolic compounds and ascorbic acid
damages nutritional value and sensory properties, leading to a
low commercial value. However, cashew apple juice is widely
consumed in Brazil and increased consumption in external mar-
kets depends on the knowledge of degradation mechanisms to
enable technological improvement of its processing (Mesquita,
Maia, Souza Filho, & Nassu, 2003).
The purpose of this study was to extract and characterise poly-
phenol oxidase from cashew apple, and to evaluate the effects of
wounding and temperature on cashew apple PPO activity, phenolic
acid composition and browning.

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.08.048
 C. Queiroz et al. / Food Chemistry 125 (2011) 128–132
2. Material and methods
2.1. Material and chemicals
Cashews (A. occidentale, L.) were harvested in Ceará, Brazil, at
commercial maturity and were acquired at market centres in Rio
de Janeiro, Brazil.
Authentic standards for catechol, catechin, cinnamic acid, gallic
acid, ferulic acid, p-coumaric acid, protocatechuic acid, vanillic
acid, syringic acid, caffeic acid, ascorbic acid and 5-hydroxymethyl-
furfural were obtained from Sigma–Aldrich, USA. Trifluoroacetic
acid (TFA) and citric acid were purchased from Merck, Germany.
All other chemicals were of analytical grade.
2.2. Sample preparation
Cashew apples were washed in distilled water: nuts were re-
moved and cashew apple juice was obtained using a juicer extrac-
tor (Samsom, USA) without water addition. All procedures were
carried out at room temperature.
2.3. Enzyme extraction and partial purification
Cashew apple juice (50 ml) was mixed with 50 ml of 10 mM so-
dium phosphate buffer (pH 6.5) to obtain a cashew apple homog-
enate, which was centrifuged at 12,000g for 30 min at 4 °C.
(NH4)2SO4 was added to the supernatant, defined as crude extract,
to obtain 95% saturation and then centrifuged at 12,000g for
30 min at 4 °C. The precipitate was dissolved in 5 ml of 10 mM
phosphate buffer (pH 6.5) and dialysed against the same buffer
at 4 °C for 24 h with four exchanges of buffer. The dialysed sample,
named enzyme extract, was used to measure PPO activity in the
following experiments.
The protein content was determined according to the dye-bind-
ing method of Bradford (1976), using bovine serum albumin as
standard.
2.4. PPO assay
PPO enzymatic activity was measured using catechol as the
exogenous substrate according to Kumar et al. (2008) with some
modifications. The reaction medium contained 200 ll of enzymatic
extract and 800 ll of 50 mM catechol in 50 mM phosphate buffer
(pH 6.5), and was incubated at 37 °C for 120 min. The blank control
sample contained 1 ml of substrate solution. PPO activity was mea-
sured by increased absorbance at 420 nm, using a spectrophotom-
eter (Beckman DU 650, USA). One unit of PPO activity was defined
as the amount of enzyme that caused a change in absorbance of
0.001 O.D. per min.
2.5. PPO enzymatic kinetics
Michaelis–Menten constant (Km) and maximum reaction
velocity (Vmax) were determined, using catechol at different con-
centrations (5, 15, 25, 35, 50, 75, 100 and 200 mM), under the con-
ditions described above.
2.6. PPO substrate specificity
Cashew apple PPO activity was measured with different sub-
strates (catechol, caffeic acid, gallic acid and catechin) at concen-
trations of 10 mM in 50 mM phosphate buffer (pH 6.5). Relative
PPO activity was described as the percentage of the activity using
catechol as substrate.
129
2.7. Optimum pH
PPO activity was determined using buffer with varied pH (2.0–
7.0). The buffers used were glycine–HCl, citrate–citric acid, Tris–
HCl and sodium acetate at 100 mM (Machado et al., 2007). The
reaction medium contained 200 ll of enzymatic extract and
800 ll of 50 mM catechol diluted in the different buffers, and
was incubated at 37 °C for 120 min.
2.8. Effect of modulating agents on PPO activity
PPO activity was tested in the presence of varied concentrations
(0.001–100 mM) of sodium chloride, sodium sulphite, sodium
metabisulphite, ascorbic acid and citric acid. PPO activity was
determined using 50 mM catechol as substrate. The control had
the same concentration of enzyme, in the absence of inhibitor.
The results were reported as relative activity (%).
2.9. Effect of processing on PPO activity and phenolic acid composition
2.9.1. General
To study the effects of injury and temperature on PPO activity
and phenolic acids composition, 1200 g of cashew apple were cut
into small pieces, approximately 1 cm3 each, and kept at 2 °C,
27 °C and 40 °C for 24 h. After this time the juice was extracted,
packaged under vacuum and stored at 22 °C until further analy-
ses. Sample control was the juice extracted immediately after the
cutting. Extraction was conducted, as described previously, and
PPO activity was measured as described. In addition, phenolic acid
composition was determined by HPLC.
2.9.2. Phenolic acid extraction and identification
Cashew apple juice (25 ml) was extracted with ethyl acetate
(25 ml) four times. The extracts were combined, filtered through
anhydrous Na2SO4 and brought to dryness in a rotary evaporator
at 40 °C under vacuum. The residue was re-dissolved in 5 ml meth-
anol:TFA (0.02%) and analysed for free phenolic acids. Conjugated
phenolic acids were extracted according to Ross, Beta, and Arnt-
field (2009). A 5 ml aliquot of cashew juice was hydrolysed in
2 M NaOH (with 1% ascorbic acid and 10 mM EDTA) and kept at
40 °C. After 30 min, the sample was allowed to cool and 1.4 ml of
7.2 M HCl was added to acidify the reaction mixture. The liberated
phenolic acids were extracted twice with ethyl acetate (6 ml). The
mixture was centrifuged for 10 min at 3000 g. The supernatants
were combined and evaporated to dryness under reduced pressure.
The residue was re-dissolved in 2 ml of methanol:water (75:25).
2.9.3. HPLC analyses
The chromatographic analyses were performed according to
Wen, Li, Di, Liao, and Liu (2005), with slight modifications, using
a low pressure gradient HPLC Shimadzu system equipped with a
photodiode array detector. Instrument control and data analyses
were carried out using a Shimadzu Class-VP Chromatography
workstation, with an octadecylsilane 5 lm 4.6  250 mm Waters
Symmetry column (Massachusetts, USA). The flow rate of the mo-
bile phase was 1 ml/min. Mobile phase A was water:TFA 0.02%, and
phase B was methanol:TFA 0.02%. The gradient conditions were as
follows: 0–5 min, 25% B; 5–10 min, 25–30% B; 10–16 min, 30–45%
B; 16–18 min, 45% B; 18–40 min, 45–80% B; 40–50 min, 80–25% B.
The detection wavelength was set to 270 nm. Authentic standards
of acids included gallic, protocatechuic, vanillic, syringic, ferulic,
caffeic, p-coumaric and cinnamic acids at concentrations of
0.02 mg/ml in methanol.
130 C. Queiroz et al. / Food Chemistry 125 (2011) 128–132

Table 1
Partial purification of polyphenol oxidase from cashew apple.
1 1 1
Purification steps Volume (ml) Total protein (mg) Total activity (U min ) Specific activity (U min mg protein ) Purification (fold) Yield (%)
Crude extract 89 34.7 13.4 0.38 1 100
Enzymatic extract 7 0.77 17.5 22.7 58.8 28

2.9.4. Non-enzymatic browning
Non-enzymatic browning in wounded cashew apple was deter-
mined by measuring the production of 5-hydroxymethylfurfural
(HMF) according to the method described by Cohen, Birk, Mann-
heim, and Saguy (1998). An aliquot of cashew apple juice (3 ml)
was homogenised with 3 ml of ethanol, and the mixture was cen-
trifuged at 12,000g for 10 min. The supernatant (1 ml) was added
to 1 ml of 734 mM trichloroacetic acid and 1 ml of 25 mM thiobar-
bituric acid. The reaction mixture was incubated at 40 °C for
50 min and the absorbance was recorded at 443 nm. A standard
curve was plotted, using HMF at concentrations varying from 2–
40 mg/l.
2.10. Statistical analysis
(18.5%), catechin (17.5%) and gallic acid (4.2%). The high affinity for
small o-diphenols, such as catechol, was also described for PPO ex-
tracted from butter lettuce (Gawlik-Dziki et al., 2008) and Barba-
dos cherry (Kumar et al., 2008).
3.4. Optimum pH
Cashew apple PPO showed maximal activity at pH 6.5 (data not
shown) and remained at approximately 63% at pH 7.0. PPO activity
was not apparent at pH above 7.0 due to the oxidation of substrate.
Similar pH values were found for PPO extracted from yacon root
(Neves & Silva, 2007) and medlar (Dincer, Colak, Aydin, Kadioglu,
& Güner, 2002). According to the literature, optimal pH for PPO
activity varies from 5.0 to 7.5 (Yoruk & Marshall, 2006).

The experiments were carried out in duplicate. One-way analy-

sis of variance (ANOVA) and Tukey’s post hoc test were used to 3.5. Effect of modulating agents
determine the significant differences using the software GraphPad
Prism v 5 (GraphPad Software, 2009). A p value <0.05 was consid- PPO activity can be regulated by the action of antioxidant
ered statistically significant. agents, enzyme inhibitors, acidulants, chelating agents or complex-
ing agents (Özoğlu & Bayındırlı, 2002). Fig. 1 shows the effects of
sodium chloride, sodium sulphite, sodium metabisulphite, ascorbic
3. Results and discussion
acid and citric acid on enzymatic extract of cashew apple.
Among the substances analysed, sodium chloride promoted PPO
3.1. PPO enzyme extraction and partial purification
activity. The inhibitory effect of sodium on PPO activity has been
described in grape (Rapeanu et al., 2006) and banana (Ünal,
To achieve optimum extraction of PPO from cashew apple, dif-
2007). However, PPO activation by sodium chloride, as observed
ferent conditions were tested (juice extraction equipment and con-
here, was also described for Fuji apple (Fan, Wang, & Zou, 2005)
centration of ammonium sulphate) (data not shown). The current
and Golden Delicious apple (Pizzocaro, Torreggiani, & Gilardi,
method, using a juicer extractor, produced the best results. Ammo-
1993). These reports described PPO activation by enzyme confor-
nium sulphate precipitation was used to obtain a PPO-enriched
mational changes, or altered protein association/dissociation due
fraction and to remove high molecular weight proteins from the
to modified ionic strength.
sample. The purification achieved was approximately 59-fold with
Ascorbic acid showed strong inhibition of PPO, causing com-
28.2% recovery of cashew apple PPO activity (Table 1).
plete enzyme inactivation, even at low concentrations. Different
PPO has been extracted from many sources in a latent form and
compounds may affect PPO activity by distinct mechanisms. Sul-
needs to be activated before activity analysis. The detergent SDS
phur-containing agents, such as sodium sulphite and metabisulph-
has been used to activate PPO (Sellés-Marchart et al., 2006). In this
ite, act on quinones as reducing agents regenerating polyphenols.
study, the presence of SDS, in concentrations ranging from 0.25 to
Acidulants change the pH of the medium and decrease enzyme
4 mM, did not alter PPO activity (data not shown).
activity (Özoğlu & Bayındırlı, 2002). Ünal (2007) demonstrated that
ascorbic acid, at 0.2 and 0.8 mM, resulted in 99% and 100% inhibi-
3.2. Enzymatic kinetics

PPO oxidises various phenolic substrates, but the affinity for Sodium Chloride
each compound depends upon the enzyme source. In this work, ki- 300 Citric Acid
netic constants were determined using different concentrations of
Residual Activity (%)

Sodium Sulfite
catechol as a synthetic substrate. Km was 18.8 mM, similar to the
250
Sodium Metabisulfite
value of 17.3 mM from blackberry (González, De Ancos, & Cano, 200 Ascorbic Acid
2000), but different from the 8.5 mM reported in Anamur banana
(Ünal, 2007) and 5.2 mM in Barbados cherry. The maximum reac- 150
tion rate (Vmax) value for cashew apple PPO was 13.6 U min 1 ml 1.
This was lower than values found in butter lettuce 100
(4081 U min 1 ml 1) reported by Gawlik-Dziki, Złotek, and Świeca
50
(2008).
0
3.3. Substrate specificity
1
01

1
5
0

5
10
50

0
5
00

0.
0.

10
2.
0.
0.

Enzymatic activity was tested using several different substrates. Concentration (mM)
Cashew apple PPO showed the highest activity with o-dihydroxy-
phenol catechol (100% of relative activity), followed by caffeic acid Fig. 1. Effects of modulation agents on enzyme activity.
 C. Queiroz et al. / Food Chemistry 125 (2011) 128–132 131

tions of banana PPO, respectively. A 100% inhibition with sodium 1.5

metabisulphite, at concentrations of 0.01 and 0.05 mM, was also
*

HMF content (mg/L)

observed.

1.0
3.6. Effect of processing on enzyme activity and phenolic acid
composition

3.6.1. General 0.5

PPO is a defence mechanism activated when the plant is sub-
mitted to stress. The effect of wounding on cashew apple PPO
activity is described in Table 2. After 24 h at 2 °C and 27 °C, PPO
activity increased 5-fold. However, PPO activity after 24 h at
40 °C increased only 2-fold, showing the negative effect of temper-
ature on enzyme activity. Zhao, Zhao, Wang, and Wang (2005) re-
lated enhanced activity of resistance-related enzymes, including
PPO in cucumber seedling, after a stress stimulus. Activation of de-
fence mechanisms also improved resistance of cucumber seedlings
to pathogen infection. Aquino-Bolaños and Mercado-Silva (2004)
observed activation of PPO extracted from jicama (Pachyrizus ero-
sus L. Urban) after mechanical damage and storage at 20 °C.
Stress-induced activity was also reported in apple and the authors
concluded that PPO activation in this fruit occurred due to post-
transduction pathways that act during plant development, and
not by a new synthesis of the enzyme (Kim et al., 2001).
3.6.2. Phenolic acid extraction and identification
Table 3 shows the compounds identified in cashew apple juice
submitted to mechanical stress. Only four phenolic acids were
identified in cashew apple juice: gallic acid, protocatechuic acid,
and cinnamic acid conjugate and free cinnamic acid. Broinizi
et al. (2007) identified nine phenolic acids in cashew apple pulp,
including gallic acid, protocatechuic acid and cinnamic acid, as
identified in this work. Michoudjehoun-Mestres et al. (2009) also
observed gallic acid in Brazilian cashew apples, but other com-
pounds identified by these authors, e.g. p-coumaric acid and ellagic
acid, have not been detected in the present study. These differ-
ences in phenolic composition can be attributed to different pre-
and post-harvest conditions, climate, soil, extraction methods
and analysis methods.
No modifications were observed at 2 °C and 27 °C but, at 40 °C,
increased levels of protocatechuic and cinnamic acid and a de-
crease in the cinnamic acid conjugate were detected. These results
indicate that high temperature is an important factor in altering
0.0
Control 2°C 27°C 40°C
Fig. 2. Hydroxymethylfurfural contents (mg/l) in cashew apple juice extracted after
0 h (control) and 24 h of storage at different temperatures.
phenolic composition by promoting hydrolysis of these bioactive
compounds.
3.6.3. Non-enzymatic browning
Browning in food products can occur by PPO-catalysed reac-
tions or non-PPO reactions. In the latter case, the basic types of
browning are Maillard reaction, caramelisation and ascorbic acid
oxidation that produce HMF. The HMF-content in cashew apple
juice was directly affected by temperature (Fig. 2). Twenty-four
hours after cutting, cashew apple stored at 40 °C presented the
highest HMF-content (p < 0.05), showing that browning is due to
HMF formation, because PPO activity is low at this temperature.
Production of HMF, observed here, can be due the oxidation of
ascorbic acid, because this vitamin is very sensitive and is present
at high concentrations in cashew apple (Damasceno, Fernandes,
Magalhães, & Brito, 2008; Lavinas et al., 2006).
In conclusion, cashew apple PPO appears to share some bio-
chemical characteristics of several plant PPOs, showing high sub-
strate affinity for catechol and optimum pH at 6.5. The most
potent enzymatic inhibitors were ascorbic acid and sodium sulph-
ite, while sodium chloride enhanced enzymatic activity. The effects
of processing on PPO activity and phenolic acid composition dem-
onstrate the importance of post-harvest treatment and handling, in
order to decrease crop loss and to enhance nutritional and sensory
characteristics.

Acknowledgements
Table 2
Polyphenol oxidase activity by wounded cashew apple.
The authors would like to thank the Fundação Carlos Chagas Fil-
1 1
Extract PPO activity (U min mg protein ) Activation ho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and
0 h (control) 0.62 – the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
24 h/2 °C 2.92 4.8 (CAPES) for financial support.
24 h/27 °C 3.33 5.4
24 h/40 °C 1.07 1.7
References

Aquino-Bolaños, E. N., & Mercado-Silva, E. (2004). Effects of polyphenol oxidase and

Table 3 peroxidase activity, phenolics and lignin content on the browning of cut jicama.
Retention times (min) of compounds identified in cashew apple juice in cashew apple Postharvest Biology and Technology, 33, 275–283.
juice extracted after 0 h (control) and 24 h of storage at different temperatures. Barreto, G. P. M., Souza, A. C. R., Azeredo, H. M. C., & Mercadante, A. Z. (2007).
Compostos bioativos em sub-produtos de castanha de caju. Alimentos e
Sample Gallic Protocatechuic Conjugate Free Nutrição, 18, 207–213.
acid acid cinnamic cinnamic Bradford, M. M. (1976). A rapid and sensitive method for the quantification of
acid acid microgram quantities of protein utilizing the principle of protein-dye binding.
Analytical Biochemistry, 72, 248–257.
Control 2.81 5.85 21.92 33.47 Brito, E. S., Araújo, M. C. P., Lin, L.-Z., & Harnly, J. (2007). Determination of the
Control after hydrolysis 3.39 5.70 ND 33.31 flavonoid components of cashew apple (Anacardium occidentale, L.) by LC-DAD-
2 °C 2.82 5.74 21.69 33.11 ESI/MS. Food Chemistry, 105, 1112–1118.
27 °C 2.79 6.02 22.74 34.05 Broinizi, P. R. B., Andrade-Wartha, E. R. S., Silva, A. M. O., Novoa, A. J. V., Torres, R. P.,
40 °C 2.78 5.62 21.73 33.17 Azeredo, H. M. C., et al. (2007). Evaluation on the antioxidant activity of
phenolic compounds naturally contained in by-products of the cashew apple
ND – not detectable. (Anacardium occidentale L.). Ciência e Tecnologia de Alimentos, 27, 902–908.
132 C. Queiroz et al. / Food Chemistry 125 (2011) 128–132
Cohen, E., Birk, Y., Mannheim, C. H., & Saguy, I. S. (1998). A rapid method to monitor
quality of apple juice during thermal processing. Food Science and Technology –
LWT, 31, 612–616.
Damasceno, L. F., Fernandes, F. A. N., Magalhães, M. M. A., & Brito, E. S. (2008). Non-
enzymatic browning in clarified cashew apple juice during thermal treatment:
Kinetics and process control. Food Chemistry, 106, 172–179.
Dincer, B., Colak, A., Aydin, N., Kadioglu, A., & Güner, S. (2002). Characterization of
polyphenoloxidase from medlar fruits (Mespilus germanica L. Rosaceae). Food
Chemistry, 77, 1–7.
Fan, M. H., Wang, M., & Zou, P. (2005). Effect of sodium chloride on the activity and
stability of polyphenol oxidase from Fuji apple. Journal of Food Biochemistry, 29,
221–230.
Gawlik-Dziki, U., Złotek, U., & Świeca, M. (2008). Characterization of polyphenol
oxidase from butter lettuce (Lactuca sativa var. capitata L.). Food Chemistry, 107,
129–135.
González, E. M., De Ancos, B., & Cano, M. P. (2000). Partial characterization of
peroxidase and polyphenol oxidase activities in blackberry fruits. Journal of
Agricultural and Food Chemistry, 48, 5459–5464.
IBGE. (2009). Instituto Brasileiro de Geografia e Estatística. <http://www.ibge.gov.br/
estadosat/temas.php?sigla=ce&tema=lavourapermanente2007>. Accessed:
30.09.09.
Kim, J. Y., Seo, Y. S., Kim, J. E., Sung, S. K., Song, K. J., An, G., et al. (2001). Two
polyphenol oxidases are differentially expressed during vegetative and
reproductive development and in response to wounding in the Fuji apple.
Plant Science, 161, 1145–1152.
Kumar, V. B. A., Mohan, T. C. K., & Murugan, K. (2008). Purification and kinetic
characterization of polyphenol oxidase from Barbados cherry (Malpighia glabra
L.). Food Chemistry, 110, 328–333.
Lavinas, F. C., Almeida, N. C., Miguel, M. A. L., Lopes, M. L. M., & Valente-Mesquita, V.
L. (2006). Study of the chemical and microbiological stability of cashew apple
juice in different storage conditions. Ciência e Tecnologia de Alimentos, 26,
875–883.
Machado, F. F., Coimbra, J. S. R., Rojas, E. E. G., Minim, L. A., Oliveira, F. C., & Sousa, R.
C. S. (2007). Solubility and density of egg white proteins: Effect of pH and saline
concentration. LWT – Food Science and Technology, 40, 1304–1307.
Mayer, A. M. (1987). Polyphenol oxidases in plants – Recent progress.
Phytochemistry, 26, 11–20.
Mesquita, P. C., Maia, G. A., Souza Filho, M. S. M., & Nassu, R. T. (2003).
Microbiological, physico-chemical and sensorial stability of cashew apples
(Anacardium occidentale L.) processed by combined methods. Ciência e
Tecnologia de Alimentos, 23, 366–369.
Michoudjehoun-Mestres, L., Souquet, J. M., Fulcrand, H., Bouchut, C., Reynes, M., &
Brillouet, J. M. (2009). Monomeric phenols of cashew apple (Anacardium
occidentale L.). Food Chemistry, 112, 851–857.
Neves, V. A., & Silva, M. A. (2007). Polyphenol oxidase from yacon roots (Smallanthus
sonchifolius). Food Chemistry, 55, 2424–2430.
Özoğlu, H., & Bayındırlı, A. (2002). Inhibition of enzymic browning in cloudy apple
juice with selected antibrowning agents. Food Control, 13, 213–221.
Pizzocaro, F., Torreggiani, D., & Gilardi, G. (1993). Inhibition of apple
polyphenoloxidase (PPO) by ascorbic acid, citric acid and sodium chloride.
Journal of Food Processing and Preservation, 17, 21–30.
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, 361–375.
Rapeanu, G., Loey, A. V., Smout, C., & Hendrickx, M. (2006). Biochemical
characterization and process stability of polyphenoloxidase extracted from
victoria grape (Vitis vifera ssp. Sativa). Food Chemistry, 94, 253–261.
Ross, K. A., Beta, T., & Arntfield, S. D. (2009). A comparative study on the phenolic
acids identified and quantified in dry beans using HPLC as affected by different
extraction and hydrolysis methods. Food Chemistry, 113, 336–344.
Sellés-Marchart, S., Casado-Vela, J., & Bru-Martínez, R. (2006). Isolation of a latent
polyphenol oxidase from loquat fruit (Eriobotrya japonica Lindl.): Kinetic
characterization and comparison with the active form. Archives of
Biochemistry and Biophysics, 446, 175–185.
Ünal, M. U. (2007). Properties of polyphenol oxidase from Anamur banana (Musa
cavendishii). Food Chemistry, 100, 909–913.
Wen, D., Li, C., Di, H., Liao, Y., & Liu, H. (2005). A universal HPLC method for the
determination of phenolic acids in compound herbal medicines. Journal of
Agricultural and Food Chemistry, 53, 6624–6629.
Yoruk, R., & Marshall, M. R. (2006). Physicochemical properties and function of plant
polyphenol oxidase: A review. Journal of Food Biochemistry, 27, 361–422.
Zhao, H.-C., Zhao, H., Wang, B.-C., & Wang, J.-B. (2005). Effect of local stress
induction on resistance-related enzymes in cucumber seedling. Colloids Surface.
B, Biointerfaces, 43, 37–42.