ARTICLE IN PRESS

LWT 40 (2007) 852–859

www.elsevier.com/locate/lwt

Peroxidase and polyphenol oxidase thermal inactivation by microwaves

in green coconut water simulated solutions
K.N. Matsuia, L.M. Granadob, P.V. de Oliveirac, C.C. Tadinia,
a
Department of Chemical Engineering, Escola Politécnica, São Paulo University, P.O. Box 61548, São Paulo, Zip code 05424-970, Brazil
b
Technology College Oswaldo Cruz, Rua Brig. Galvão, 540, São Paulo, Zip code 01151-000, Brazil
c
Chemistry Institute, São Paulo University, P.O. Box 26077, São Paulo, Zip code 05513-970, Brazil
Received 31 October 2005; received in revised form 20 March 2006; accepted 20 March 2006

Abstract

Enzymes from coconut water such as peroxidase (POD) and polyphenol oxidase (PPO) when in contact with oxygen begin reactions
causing nutritional and color losses. Solutions simulating the chemical constituents of coconut water were submitted to a batch process in
a microwave oven. PPO and POD inactivation data could be characterized by: PPO/water D93 1C ¼ 16.5 s (z ¼ 35.5 1C); PPO/sugars
D91 1C ¼ 18 s (z ¼ 331C); POD/water D91.5 1C ¼ 44 s (z ¼ 24 1C) and POD/sugars D92 1C ¼ 20.5 s (z ¼ 19.5 1C). The contact between salts
and enzymes promoted a drastic reduction of the initial activity. After the incidence of microwave energy at temperatures above 90 1C,
enzymes activity was not detected. These results can indicate an adequate choice of temperature conditions to inactivate coconut water
enzymes. The knowledge of how green coconut water constituents influence POD and PPO activity will supply useful information about
microwave processing of coconut water.
r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Microwaves; Peroxidase; Polyphenol oxidase; Thermal processing

1. Introduction potentially improve retention of thermolabile constituents

Green coconut water can be considered a natural
isotonic drink, due to its mineral and sugar content. It is
a very popular drink in Brazil and can be found either in
natura or processed. In order to avoid spoilage and
enzymatic browning caused by peroxidase (POD) and
polyphenol oxidase (PPO) when the product is exposed to
air for a long time, coconut water can undergo different
processes such as ultra-high temperature (UHT), conven-
tional pasteurization, refrigeration and freezing (Abreu &
Rosa, 2000; Campos, Souza, Coelho, & Glória, 1996;
Duarte, Coelho, & Leite, 2002).
Microwave heating as an alternative method for liquid
food pasteurization has gained acceptance because it offers
several advantages over the conventional method. Micro-
waves are able to heat products internally, have greater
penetration depth and faster heating rates that would
Corresponding author. Tel.: +55 11 30912258; fax: +55 11 30912255.
E-mail address: catadini@usp.br (C.C. Tadini).
in the food (Cañumir, Celis, de Bruijn, & Vidal, 2002;
Deng, Singh, & Lee, 2003; Gerard & Roberts, 2004;
Heddleson & Doores, 1994; Nikdel, Chen, Parish, Mack-
ellar, & Friedrich, 1993).
Microwave energy induces thermal effects over micro-
organisms and enzymes similar to those of conventional
heating mechanisms (Cañumir et al., 2002). However, a
problem that has often been encountered is the occurrence
of temperature profiles within a product. The measurement
of temperature profiles during microwave heating is
conducted using fiber optic probes that could be easily
incorporated into the process without disturbing it (Deng
et al., 2003; Gerard & Roberts, 2004; Nott & Hall, 1999).
Pasteurization involving enzyme inactivation by micro-
wave energy has not been commonly studied and there are
few reports on kinetic data for POD and PPO inactivation.
(Soysal & Soylemez, 2005; Tajchakavit & Ramaswamy,
1997).
POD and PPO are widely detected in many fruits and
vegetables and are closely linked to enzymatic color

0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2006.03.019
 ARTICLE IN PRESS
K.N. Matsui et al. / LWT 40 (2007) 852–859
changes with consequent loss of sensorial properties and
nutritional quality (Duarte et al., 2002; Robinson, 1991).
Different names have been associated with PPO including
tyrosinase, cresolase, cathecolase and phenolase and
generally reflect the ability of this enzyme to utilize many
different phenolic compounds as substrates. POD is a
group of enzymes that catalyses oxidation reactions
reducing hydrogen peroxide to water while oxidizing a
variety of substrates (Robinson, 1991).
PPO and POD are very resistant to heat and therefore
are considered biological indicators of thermal process-
ing (Robinson, 1991; Weng, Hendrickx, Maesmans, &
Tobback, 1991).
The aim of this work was to determine POD and PPO
inactivation by microwave heating in coconut water
simulated solutions and to verify possible influences of
the major chemical constituents in coconut water on
enzyme activities.
2. Materials and methods
2.1. Simulated solutions
The solutions PPO/sugars, POD/sugars, PPO/salts,
POD/salts, PPO/salts/sugars and POD/salts/sugars were
prepared with sugars and salts concentrations similar to
average contents of green coconut water reported in
literature (Aleixo, Nóbrega, Santos Júnior, & Muller,
2000; Santoso, Kubo, Ota, Tadokoro, & Maekawa, 1996).
Commercial horseradish POD (Sigma-P6140) and mush-
room Tyrosinase (Sigma-T3824) were used to prepare the
enzyme stock in distilled water: 1.7  104 g PPO/100 ml
and 7.8  104 g POD/100 ml and maintained under freezing
conditions at 80 1C. Simulated solutions were prepared as
following: 1 ml of enzyme stock was diluted in 100 ml of
distilled water to obtain PPO/water solution; 1 ml of
enzyme stock was diluted in 100 ml of sugars solution
containing 0.28 g of sucrose, 2.38 g of glucose and 2.40 g of
fructose to obtain PPO/sugars solution; 1 ml of enzyme
stock was diluted in 100 ml of salts solution containing
44 mg of KH2PO4, 336 mg of K2SO4, 31 mg of Na2SO4,
47 mg of CaCl2 and 20 mg of MgCl2 to obtain PPO/salts
solution; 1 ml of enzyme stock was diluted in 100 ml of
sugars and salts solution containing 0.28 g of sucrose,
2.38 g of glucose, 2.40 g of fructose, 44 mg of KH2PO4,
336 mg of K2SO4, 31 mg of Na2SO4, 47 mg of CaCl2 and
20 mg of MgCl2 to obtain PPO/sugars/salts solution. The
same procedure was done to POD.
2.2. Microwave thermal treatment
The microwave oven (model star system, CEM Corpora-
tion, Matthews, USA) at 2450 MHz contains two cavities
where specific glass tubes (310 mm length and 41 mm
diameter) were inserted and an automatic program that
controls microwaves incidence. Maximum volume of 20 ml
was used to obtain homogeneity of microwaves incidence.
853
Samples of simulated solutions were individually submitted
to a batch process in the microwave oven at different
temperatures in the 60–100 1C.
Real temperature–time profiles during batch processing
were acquired using an optic fiber probe, calibrated with
distilled water using a calibrated thermometer model
TRB (Gavea Sensors, Rio de Janeiro, Brazil) inserted
centrally inside the glass tube. Temperature readings
were recorded using a continuous data acquisition system.
After microwaves incidence, the glass tube was removed
from the microwave oven and inserted into an ice bath
to accelerate cooling. Subsequently 2 ml samples were
collected to determine the enzymatic activity and were
quickly cooled in liquid nitrogen and kept in a freezer at
80 1C, model MDF-U3086S (Sanyo Electric Co. Ltd.,
Japan).
2.3. Determination of enzymatic activity
POD activity was monitored at 405 nm in a spectro-
photometer UV–VIS, model 700 PLUS (Femto, São Paulo,
Brazil) according to the method described by Pütter and
Becker (1983). A test tube containing 7.0 ml of buffer
(Na2HPO4  2H2O+KH2PO4) pH 6.0 and 0.8 ml of ABTS
(2.2 azino-bis 3-ethylbenzthiazoline-6-sulfonic acid) solu-
tion (2  102 mole/l) and 0.8 ml of hydrogen peroxide
solution (0.1% v/v), was immersed in a controlled
temperature bath model U2C (Veb MLW, Saxony,
Germany) at 25 1C, for 5 min for thermal stabilization.
After that 1.0 ml aliquot of simulated solution was added
to this solution. The reference value of POD (0.000
absorbance) was determined using a blank solution
containing ABTS and hydrogen peroxide.
PPO activity was monitored spectrophotometrically at
425 nm, according to the method described by Campos
et al. (1996). A test tube containing 5.5 ml of 0.2 mole/l
sodium phosphate buffer (pH 6.0) and 1.5 ml of 0.2 mole/l
pyrocatechol solution (15890, Fluka) was immersed in
a controlled temperature bath at 25 1C, for 5 min for
thermal stabilization. After that 1.0 ml aliquot of simulated
solution was added to this solution. The reference value
of PPO (0.000 absorbance) was determined using a test
tube containing 5.5 ml of 0.2 mole/l sodium phosphate
buffer (pH 6.0) and 1.5 ml of 0.2 mole/l pyrocatechol
solution.
For both enzymes the absorbance was acquired every
10 s during 30 min. The data obtained was plotted against
time and the PPO and POD activity was calculated from
the slope of the initial linear portion of the curve.
All enzyme activities were analysed in duplicate. In both
cases, one unit of enzyme activity was defined as the
quantity necessary to produce an increase in absorbance of
0.001 per ml of sample per second.
The residual activity was determined as (A/A0) where:
A ¼ mean enzyme activity (after microwave heating);
A0 ¼ mean initial enzyme activity (before microwave
heating).
 ARTICLE IN PRESS
854 K.N. Matsui et al. / LWT 40 (2007) 852–859

2.4. Physical chemical analyses 90

80
Total acidity and soluble solids were determined
70
according to AOAC methods. Total acidity was expressed

Temperature (°C)
as malic acid percentage. Titration was carried out in the 60
pH-Stat PHM-290 (Radiometer Analytical S.A., Lyon, 50
France), until pH 8.2 was reached (Association of Official
40
Analytical Chemists (AOAC), 1995).
Soluble solids, expressed as 1Brix were determined for a 30
portable refractometer and corrected by temperature 20
(AOAC, 1995).
10
The pH was directly measured using the pH-Stat PHM- 0 50 100 150 200 250 300 350 400 450
290 (Radiometer Analytical S.A., Lyon, France) (AOAC, (a) Time (s)
1995).
90

2.5. Kinetic data analysis: nonisothermal heating conditions 80

70
In most thermal processing situations, food products are

Temperature (°C)
subjected to nonisothermal heating conditions. The accu- 60
mulated lethality (L) is obtained by integration of the lethal 50
effects of the temperature profile during the come-up, hold
40
and cooling periods using the relationship:
Z t 30
L¼ 10ðTT ref Þ=z dt. (1)
0 20

Computation of the lethality requires data on the z value 10

that can be obtained from a regression of log D value vs.
temperature and Tref is the reference temperature. In batch
microwave heating there is no isothermal hold period. First
estimates of D values can be calculated based on the total
residence time of the product in the microwave cavity, and
from D values a first estimate of z value is obtained.
Correction of heating times and calculation of D and z can
then be repeated to get convergence of D and z
values (Tajchakavit & Ramaswamy, 1997; Tajchakavit,
Ramaswamy, & Fustier, 1998; Toledo, 1991).
According to Tajchakavit and Ramaswamy (1997), since
there is no specific isothermal hold period, any temperature
within the range of study could be used as a reference
temperature.
The experiments were organized in groups according to
maximum temperature (Tmax), and for each group the
highest temperature was considered as reference tempera-
ture (Tref). This allowed the determination of the equiva-
lent time (tequiv) and kinetic parameters (D and z). This
procedure was applied for both enzymes (PPO and POD)
for all simulated solutions. A z value of 15 1C was
considered the initial value used to calculate the first
equivalent time.
3. Results and discussion
3.1. Time–temperature profiles
PPO and POD simulated solutions were tested at
different temperature conditions. Fig. 1 shows typical
temperature profiles obtained for PPO and POD simulated
0 50 100 150 200 250 300 350 400 450
(b) Time (s)
Fig. 1. Temperature–time profiles for PPO simulated solutions (a) [(m)
PPO/water; (’) PPO/salts; (&) PPO/sugars; (J) PPO/salts/sugars] and
for POD simulated solutions (b) [(m) POD/water; (’) POD/salts; (&)
POD/sugars; (J) POD/salts/sugars] submitted to microwave heating.
solutions at the same conditions of microwave incidence.
The direct contact between the optic fiber probe and the
simulated solutions allowed real acquisition of temperature
data.
It was observed that the temperature–time profiles of
PPO and POD solutions have similar behaviors. Solutions
with sugars and water reached about 75 1C while solutions
containing salts (PPO/salts, PPO/salts/sugars, POD/salts
and POD/salts/sugars) reached higher temperatures up to
85 1C. These results were expected since ionized compo-
nents collide randomly with ionized and nonionized
molecules when submitted to an electromagnetic field,
causing heat generation by friction. Ionic movement is one
of the most important mechanisms that contribute to
convert electromagnetic energy into heat (Heddleson &
Doores, 1994; Mudgett, 1986).
3.2. Polyphenol oxidase microwave inactivation kinetics
Table 1 presents the maximum temperature (Tmax) of
each condition, reference temperature (Tref), equivalent
heating time (tequiv) calculated based on accumulated
lethality (Eq. (1)) and D value for PPO/water, PPO/sugars,
 ARTICLE IN PRESS
K.N. Matsui et al. / LWT 40 (2007) 852–859 855

Table 1
Maximum temperature (Tmax), reference temperature (Tref), equivalent heating time (tequiv) and D value for PPO simulated solutions submitted to
microwaves

PPO simulated solutions

Watera Sugarsb Saltsc Salts/sugarsd

Tmax Tref tequiv (s) D* (s) Tmax Tref tequiv (s) D* (s) Tmax Tref tequiv (s) Dl* (s) Tmax Tref tequiv (s) Dl* (s)
(1C) (1C) (1C) (1C) (1C) (1C) (1C) (1C)

74.1 76.5 78.7 48 74.4 75.6 68.3 51 64.3 73.4 11.3 10 70.1 75.7 16.9 18
76.5 74.4 75.6 86.1 69.1 19.0 72.9 30.3
71.7 33.2 75.7 42.9
81.0 82.1 70.0 33 78.3 79.8 76.1 41 73.4 44.4
82.0 81.7 79.8 83.0
82.1 85.4 80.8 89.6 14.2 14 81.9 85.7 26.7 20
82.6 84.3 63.5 26 84.1 22.6 83.4 41.8
84.6 86.8 66.6 27 83.0 75.1 87.1 32.7 85.7 50.8
85.3 71.0 83.1 67.5 89.6 38.3
86.8 72.6 84.3 70.4
97.4 99.2 46.0 — 96.2 98.7 36.2 —
90.9 92.9 44.6 17 90.0 91.1 50.4 18 97.5 55.7 96.5 39.1
91.7 44.9 91.1 46.6 98.5 22.8 97.2 19.7
92.9 45.8 99.2 29.9 98.7 26.1

*Determined according to Fig. 2.Dl ¼ thermo labile fraction.

a
pH 6.770.1 and (0.02670.005) % malic acid.
b
pH 6.570.2, (0.02770.007) % malic acid and (4.5370.15) 1Brix.
c
pH 5.070.0 and (0.06570.004) % malic acid
d
pH 5.070.1, (0.06870.005) % malic acid and (4.8670.38) 1Brix.

2 2

1.5 1.5
Log (A/Ao x100)

Log (A/Ao x100)

1 1

0.5 0.5

0 0

-0.5 -0.5

-1 -1
0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90
(a) Equivalent time (s) (c) Equivalent time (s)

1000 1000

100 100
D-value (s)

D-value (s)

10 10

1 1
60 70 80 90 100 110 60 70 80 90 100
(b) Temperature (°C) (d) Temperature (°C)

Fig. 2. Residual PPO activity in aqueous solution (a) [(m) 76.5 1C; (S) 82.1 1C; (J) 86.8 1C;(E) 92.9 1C]; and in sugars solution (c) [(  ) 75.6 1C; (’)
79.8 1C; (K) 84.3 1C; (B) 91.1 1C] according to equivalent time at different temperatures when submitted to heating by microwaves; Temperature
sensitivity curves of PPO/water (b) [(n) z ¼ 35.5 1C and R2 ¼ 0.99] and PPO/sugars (d) [(&) z ¼ 33 1C and R2 ¼ 0.98].
 ARTICLE IN PRESS
856 K.N. Matsui et al. / LWT 40 (2007) 852–859

PPO/salts and PPO/salts/sugars simulated solutions sub- 2

mitted to microwaves heating.
Fig. 2 shows the residual PPO activity in aqueous and in 1.5
sugar simulated solutions as a function of equivalent

Log (A/Ao x100)

heating time at various temperatures under microwave 1
heating conditions and the regression of residual activity
vs. corrected equivalent time originated the D values by 0.5
iterative method. The log-linear decrease of enzyme activity
as a function of equivalent heating time showed that for 0
temperatures close to 80 1C up to two log reductions
occurred, while above this temperature almost three log
-0.5
reductions were reached. 0 10 20 30 40 50
The z values for PPO/water and PPO/sugar solutions (a) Equivalent time (s)
were similar D93 1C ¼ 16.5 s (z ¼ 35.5 1C) and D91 1C ¼ 18 s
2
(z ¼ 33 1C), respectively, as presented in Fig. 2. Weemaes et
al. (1997) determined the kinetic parameters (D and z
1.5
values) for mushroom PPO when submitted to conven-

Log (A/Ao x100)

tional thermal processing. The results show irreversible
first-order inactivation. A batch with enzyme units similar 1

of this work was tested and the D53 1C value obtained was
55 min (z ¼ 6.521C). 0.5
Campos et al. (1996) determined PPO residual activity in
coconut water heated in a water-bath. Inactivations above 0
90%were only encountered for temperatures above 90 1C
held for more than 90 s. -0.5
0 10 20 30 40 50 60
Fig. 3 shows the inactivation curves of PPO/salts and (b) Equivalent time (s)
PPO/salts/sugars solutions as a function of equivalent
heating time under microwave heating conditions. It can Fig. 3. Residual PPO activity in salts solution (a) [(J) 73.4 1C; (’)
89.6 1C] and in salts/sugars solutions (b) [(E) 75.7 1C; (n) 85.7 1C]
be observed in these cases, two first-order rate curves
according to equivalent time at different temperatures when submitted to
denoting a thermoresistant fraction in these temperatures. heating by microwaves.
The kinetic parameter of the thermolabile fraction can
be obtained from the slope of first linear section of the
curve. 1.4
After microwaves incidence, PPO residual activity of the
solution with salts at Tref ¼ 99.2 1C and of the solution 1.2
with salts/sugars at Tref ¼ 98.7 1C was not detected. Even 1
though the same amount of enzymatic extract was added in
Abs at 425 nm

all simulated solutions, results showed that initial PPO/ 0.8

salts activity was lower than for the other solutions.
Campos et al. (1996), Duangmal and Apenten (1999), 0.6
Mendonc- a and Guerra (2003), Zawistowsky, Biliaderis,
0.4
and Eskin (1991) observed that in presence of salts there
was a significant decrease in enzyme thermostability; this is 0.2
due to salt-induced changes in enzyme conformation and
possibly the dissociation of thermostable aggregated 0
molecules. 0 200 400 600 800 1000 1200
Time (s)
Catechol solution, even without the presence of PPO,
presented an increase in absorbance. Exposure to light for Fig. 4. Initial PPO activity for each simulated solution in comparison to
a certain time period causes substrate oxidation and catechol oxidation [(&) PPO/water; (K) PPO/salts; (m) PPO/sugars; (n)
consequent darkening; this fact could result in over- PPO/salts/sugars; (J) catechol oxidation].
estimation of the residual PPO/salts activity after micro-
wave incidence and suggest a false interpretation of the
results. on enzyme denaturation and the efficiency of thermal
To minimize the catechol oxidation interference in this microwave processing could then be determined.
study, higher PPO concentrations than those found in Absorbance vs. time readings for solutions containing
natural coconut water were tested, because an increase in PPO demonstrate that the salts considerably reduced the
PPO activity would probably reduce the effect of the salts enzymes thermostability. Comparing initial activity of the
 ARTICLE IN PRESS
K.N. Matsui et al. / LWT 40 (2007) 852–859 857

enzymes it can be observed in Fig. 4, that even though the
same amount of enzyme extract was added to all the
solutions, PPO/salts and PPO/salts/sugars presented lower
initial activities, particularly the PPO/salts solutions. This
decrease in the enzyme’s initial activity renders determina-
tion of the residual activity after microwave incidence
impossible, since the reaction rate is then so low that the
change in absorbance falls within the precision range of the
equipment, causing significant errors in the results.
3.3. Peroxidase microwave inactivation kinetics
Table 2 presents the maximum temperature (Tmax) of
each condition, reference temperature (Tref), equivalent
heating time (tequiv) calculated based on accumulated
lethality (Eq. (1)) and D value for POD/water, POD/
sugars, POD/salts and POD/salts/sugars simulated solu-
tions submitted to microwave heating.
POD/water solution presented a slight decrease in
activity when submitted to microwaves heating at the
studied temperatures (Fig. 5). At similar process condi-
tions, POD/sugars solutions presented higher activity
decrease. The sugars that were added to the solution
contributed to decrease POD activity.
Gibriel, El-Sahrigi, Kandil, and El-Mansy (1978)
showed that the presence of sucrose in the reaction mixture
inhibited POD activity in apricot. Chang, Park, and Lund
(1988) showed by differential scanning calorimetry that in
the presence of 10% sucrose POD thermal stability was
reduced. For a range of sugars tested, fructose was the
most effective in reducing the enzyme thermostability and
it was suggested that this was due to the interaction of
fructose with the protein amino acids. Once more, the
equipment did not allow data at different exposure times to
be obtained, so the inactivation behavior of POD could not
be determined.
The D and z values encountered for POD/water and
POD/sugars solutions were D91.5 1C ¼ 44 s (z ¼ 24 1C) and
D92 1C ¼ 20.5 s (z ¼ 19.5 1C), respectively, similar to those
reported in the literature.
Weng et al. (1991) reported a z value of 26.371.8 1C for
horseradish peroxidase (HRP) in aqueous solution and
they found biphasic behavior of the heat inactivation
when submitted to conventional heat treatment. Joffe
and Ball (1962) obtained a z value of 27.7 1C for HRP
in the temperature range 85–150 1C. Ling and Lund
(1978) reported a D82 1C ¼ 1.2 min (z ¼ 17 1C) for heat-
labile and D82 1C ¼ 42 min (z ¼ 27.3 1C) for heat-stable
HRP.
The carrot POD activity was determined by Soysal and
Soylemez (2005) and its inactivation was studied by
thermal and microwave heating. Biphasic behavior of
enzyme inactivation was observed for the microwave
treatment at low microwave power and monophasic
behavior was observed at high microwave power.
The POD/salts solution did not present significant
activity decrease for Tref ¼ 62.7 and 85.9 1C. For
Tmax ¼ 95.2 and 95.4 1C, no residual activity was detected.
The POD in the salts/sugars solution presented for
Tref ¼ 69.4 and 80.9 1C activity decrease less than one log
cycle, while for Tref ¼ 90.7 1C there was a one log cycle
decrease and for Tref ¼ 96.4 1C no residual activity was
detected. For all studied solutions, POD was more resistant
at temperatures below 90 1C than PPO.

Table 2
Maximum temperature (Tmax), reference temperature (Tref), equivalent heating time (tequiv) and D value for POD simulated solutions submitted to
microwaves

POD simulated solutions

Watera Sugarsb Saltsc Salts/sugarsd

Tmax Tref tequiv (s) D* (s) Tmax Tref(1C) tequiv (s) D* (s) Tmax Tref t equiv D* (s) Tmax Tref tequiv (s) D* (s)
(1C) (1C) (1C) (1C) (1C) (s) (1C) (1C)

76.4 77.4 57.9 167 67.0 75.8 15.6 144 57.1 62.7 18.8 — 61.9 69.4 14.2 —
77.4 61.6 68.0 17.9 62.7 37.3 69.4 38.4
75.4 54.4
81.5 83.9 41.4 93 75.8 48.9 72.2 85.9 5.2 — 75.1 80.9 18.5 —
83.9 57.7 77.1 10.7 80.9 45.1
81.0 82.3 50.8 54 80.3 17.2
82.3 50.9 85.9 49.9 83.5 90.7 16.0 —
88.5 91.5 27.9 44 84.0 19.3
91.5 31.1 90.6 91.9 31.4 21 95.2 95.4 56.1 — 90.7 50.3
91.9 24.2 95.4 51.4
95.1 96.4 69.1 —
96.4 48.5

*Determined according to Fig. 5.

a
pH 6.270.1 and (0.02470.003) % malic acid.
b
pH 6.370.1, (0.02170.004) % malic acid and (4.5370.22) 1Brix.
c
pH 5.070.0 and (0.04870.002) % malic acid
d
pH 5.270.1, (0.06870.005) % malic acid and (4.9370.14) 1Brix.
 ARTICLE IN PRESS
858 K.N. Matsui et al. / LWT 40 (2007) 852–859

2 2

1.5
Log (A/Ao x100)

Log (A/Ao x100)

1.5

1
0.5

0.5 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60
(a) Equivalent time (s) (c) Equivalent time (s)

1000 1000

100 100

D value (s)
D-value (s)

10 10

1 1
70 80 90 100 60 70 80 90 100
(b) Temperature (°C) (d) Temperature (°C)

Fig. 5. Residual POD activity in aqueous solution (a) [(E) 77.4 1C; (J) 83.9 1C; (’) 91.51C]; and in sugars solution (c) [(m) 75.8 1C; (J) 82.3 1C; (E)
91.9 1C] according to equivalent time at different temperatures when submitted to heating by microwaves; temperature sensitivity curves of POD/water (b)
[(K) z ¼ 24 1C and R2 ¼ 0.99] and POD/sugars (d) [(’) z ¼ 19.5 1C and R2 ¼ 0.99].

POD/salts and POD/salts/sugars solutions presented the Acknowledgements

same behavior observed for PPO solutions, presenting
lower initial activities demonstrating that the salts also
affect POD activity. The activity of POD/salts and POD/
salts/sugars solutions treated at temperatures above 90 1C
was not detected, due to the combined effect produced by
the salts and microwaves. Assays with ABTS were under-
taken but no change in absorbance was detected.
The authors wish to thank FAPESP (The State of São
Paulo Research Foundation) for the research grant and
Gavea Sensors Measurement Solutions Ltd. for technical
support. P.V. de Oliveira is also thankful to The National
Council for Scientific and Technological Development
(CNPq) by the research ship provided.

4. Conclusions
Focused-microwave oven showed to be a good alter-
native for enzyme inactivation studies using microwave
heating. The optic fiber probe allowed reliable tempera-
ture–time profiles by microwave heat processing.
Kinetic parameters (D and z) were estimated for PPO/
water and PPO/sugars and presented similar behavior. The
activity of PPO in both solutions was considerably reduced
when they were submitted to microwave heating.
Sugars influenced POD more than PPO inactivation.
Even so, POD was more resistant to microwave heating for
all the green coconut water simulated solutions studied.
The salts significantly affected PPO and POD stability.
At temperatures above 90 1C, the combined effect of salts
and microwave energy reduced PPO and POD enzymatic
activity to undetectable levels.
These results can indicate an adequate choice tempera-
ture conditions to inactivate green coconut water enzymes.
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