* Manuscript Click here to view linked References
Thermal inactivation kinetics of vegetable peroxidases
1 2
Halina Połata, Alina Wilińska, Jolanta Bryjak, Milan Polakovič*
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Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental
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Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology,
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Radlinského 9, 812 37 Bratislava, Slovakia
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Abs tract
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Thermal stability of peroxidases present in raw vegetable mixtures was investigated in order
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to identify adequate mechanisms and corresponding kinetic models of inactivation. Inactivation
13
experiments were carried out for each material at five different temperatures which were from the
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range 58–74 ºC for broccoli and potato juices and 62–78 ºC for carrot juice. Using the
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multitemperature evaluation of inactivation data, a simple isozyme model was verified for the
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inactivation of broccoli peroxidase. A combined three-reaction mechanism, which assumed
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simple irreversible inactivation for one isoform and Lumry-Eyring mechanism for the other one,
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was identified for carrot and potato peroxidases.
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*
Corresponding author: : Phone: + 421 2 59325254, Fax: + 421 2 52496920,
[email protected]
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Keywords: peroxidase, thermal processing, vegetable juice, enzyme stability, inactivation
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kinetics, multitemperature modelling.
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1. Introductio n
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Consumers are interested in thermally processed food in which important nutritive compounds
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are damaged as little as possible. Therefore it is of great importance to specify proper conditions for
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food sterilization. For example, heat treatment of fruit and vegetable products should assure their
29
microbiological safety, prevent browning and loss of colour, and simultaneously, it should not
30
affect their natural qualities. To accomplish these requirements, a comprehensive study of thermal
31
processing must be made.
32
Peroxidases (POD's) are used as blanching indicators since they belong to the most stable and
33
widespread plant enzymes and have a certain effect on the loss of colour and textural changes of
34
fruits and vegetables (Yemenicioğlu, Özkan, Velioğlu, & Cemeroğlu, 1998, Forsyth, Owusu
35
Apenten, & Robinson, 1999; Icier, Yildiz, H., & Baysal, 2006). Their advantage compared to other
36
potential blanching index enzymes is a simple, inexpensive activity measurement (Khan &
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Robinson, 1993b; Tijskens, Rodis, Hertog, Waldron, Ingham, Proxenia, & van Dijk, 1997;
38
Yemenicioğlu et al., 1998; Forsyth et al., 1999; Icier et al., 2006;). For example, horseradish
39
peroxidase was used for the development of time-temperature integrators that are systems
40
simulating temperature resistance of target microorganisms in thermal food processing (Lemos,
41
Oliveira, & Saraiva, 2000).
2
42
POD is a monomeric, glycosylated protein containing haem as prosthetic group (McLellan &
43
Robinson, 1987; Khan & Robinson, 1993a, 1993b; Yang, Gray, & Montgomery, 1996; Tijskens et
44
al., 1997; Forsyth & Robinson, 1998; Forsyth et al., 1999; Leon, Alpeeva, Chubar, Galaev,
45
Csoregi, & Sakharov, 2002; Carvalho, Melo, Ferreira, Neves-Petersen, Petersen, & Aires-Barros,
46
2003; Wang, Burhenne, Kristensen, & Rasmussen, 2004; Johri, Jamwal, Rasool, Kumar, Verma,
47
& Qazi, 2005). POD occurs in plant cells in both soluble and ionically bound isoforms that have
48
different molecular masses, pI (Khan & Robinson, 1993a; Forsyth & Robinson, 1998; Johri et al.,
49
2005), substrate specifity (Khan & Robinson, 1994) and thermal stability ( McLellan & Robinson,
50
1987; Khan & Robinson, 1993b; Yemenicioğlu et al., 1998; Forsyth et al., 1999; Johri et al., 2005).
51
The amount of covalently bounded carbohydrates significantly differ for POD isoforms or POD
52
from different sources (Yang et al., 1996). The primary function of POD in plants is the reduction
53
of hydrogen peroxide at the expense of oxidation of phenolic compounds. It is responsible for the
54
mechanical properties of cell walls during extension, cell adhesion and disease resistance (Tijskens
55
et al., 1997). The kinetics of POD catalytic action as well as its substrate specificity was widely
56
studied (Khan & Robinson, 1993a, 1994; Forsyth & Robinson, 1998; Leon et al., 2002; Rani &
57
Abraham; Kamal & Behere, 2003; Santos de Araujo, Omena de Oliveira, Salgueiro Machado, &
58
Pletsch, 2004; Johri et al., 2005).
59
POD thermal inactivation was studied for purified isoperoxidases (Khan & Robinson, 1993b;
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Forsyth et al., 1999; Lemos et al., 2000; Machado & Saraiva, 2002; Carvalho et al., 2003; Kamal &
61
Behere, 2003), in crude plant extracts (Khan & Robinson, 1993b; Yemenicioğlu et al., 1998;
62
Quitão-Teixeira, Aguiló-Aguayo, Ramos, & Martín-Belloso, 2008; Rudra, Shivhare, Basu, &
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Sarkar, 2007) and in vegetable particles (Icier et al., 2006). Different inactivation mechanisms were
64
identified. Khan and Robinson (1993b) found that even the inactivation mechanisms of highly
65
purified isoforms are complex. They suggested a micro-heterogeneity of purified isozymes to be 3
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responsible for producing non-first-order inactivation plots. The origin of the heterogeneity was
67
assigned to different moieties of covalently bound neutral carbohydrates. Machado and Saraiva
68
(2002) found a biexponential model that described the inactivation kinetics of horseradish POD
69
well but was not associated with any exact mechanism.
70
An extensive study of thermal inactivation of an anionic horseradish POD was performed
71
using differential scanning calorimetry, circular dichroism and tryptophan fluorescence (Carvalho
72
et al., 2003). All three methods confirmed a hypothesis that the enzyme inactivation was a two-
73
step process governed by the Lumry-Eyring mechanism. It was found that an inactive
74
intermediate in distinction to a final, irreversibly denatured form was capable to incorporate a
75
haem. Another type of series inactivation mechanism assuming partially inactivated intermediate
76
species was postulated by Forsyth et al. (1999). Tijskens et al. (1997) who studied POD
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inactivation in slices of peaches, carrots and potatoes observed different behaviour of bound and
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soluble isoforms of the enzyme each characterized by first-order kinetics. Moreover, the bound
79
form underwent a transition into the soluble form.
80
Conventional heating was applied in this study in order to examine the inactivation of POD in
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broccoli, carrot and potato purees. The objective was to identify suitable mechanisms of POD
82
inactivation and to obtain kinetic parameters that can be used in further analyses.
83 84 85
2. M aterial s a nd meth ods
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2.1. Vegetable juices
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Four vegetable mixtures based on smashed carrot, broccoli, potatoes and potatoes with
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spring onion were prepared according to the recipes provided by Nature’s Best (Drogheda,
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Ireland). The mixtures contained about 97 % of vegetable components. Their exact composition
92
is given in Table 1. Vegetables, butter, salt and pepper were purchased in local shops and fresh
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milk was delivered by a dairy company (Rajo, Bratislava, Slovakia). Broccoli was washed and
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cut, whereas carrot and potatoes were first peeled. All ingredients of the mixtures were mixed,
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chopped with a blender and squeezed in a juice extractor. The juices obtained were portioned out
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into amounts needed for one experiment and frozen. The juices were defrosted at room
97
temperature before their use in inactivation experiments.
98 99
2.2. I n a c t i v a t i o n e x p e r i m e n t s
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Inactivation experiments were performed in 1.5 ml plastic test tubes that were pre-
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incubated in a water bath at an inactivation temperature and then filled with 0.8 ml of vegetable
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juice of ambient temperature. In specified time intervals, the tubes were taken out from the bath,
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immediately cooled down for 5 minutes in an ice-water/ethanol mixture (-4 ºC) and then kept in
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an ice-water bath until activity measurement. Potato and carrot juice samples were centrifuged at
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12 000 for 15 minutes whereas broccoli juice samples were centrifuged at 14 000 rpm for 40
107
minutes. The supernatants were used for the determination of activity.
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In order to determine the reproducibility error of the inactivation experiments, the whole
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experiment of inactivation of broccoli POD at 66 ºC was duplicated and samples were taken in
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the same times. The variance of measured relative activity was first calculated for each time and
5
111
the mean over all time values was then obtained. The square root of the mean variance,
112
reproducibility error of relative activity, was 1.80 with 15 degrees of freedom.
113 114
2.3. Determination of POD activity
115 116
The POD activity was determined at 25 ºC. A sample with a volume of either 20 μl (carrot
117
juice supernatant) or 70 μl (potato and broccoli juice supernatants) was added to 1.45 ml of
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0.19 mM 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS) in
119
100 mM Na-acetate buffer at pH 5.5. The reaction was initiated by adding 50 μl of 0.02% (carrot
120
and broccoli juice supernatants) or 0.2 % (potato juice supernatant) H2O2. The sample volume
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and H2O2 concentration depended on the specific POD activity in different vegetables (Leon et
122
al., 2002; Wang et al., 2004). The increase of absorbance at 405 nm (Wang et al., 2004) was
123
recorded for 1–15 minutes using Cecil 9000 spectrophotometer (Cecil Instruments, Cambridge,
124
U.K.). The activity was calculated from the slope of the absorbance vs. time dependence. The
125
enzyme activity of 1 U corresponds to the rate of absorbance change of 0.001 min-1 (Mdluli,
126
2005).
127 128
2.4. H e a t t r a n s f e r e x p e r i m e n t s
129 130
Since the set inactivation temperature was reached in the entire sample only with a time
131
delay, the analysis of sample thermal history was made and the values of the heat coefficients
132
were estimated. Pre-incubated test tubes were filled with 0.8 ml of a sample and the temperature
133
was recorded every 3 seconds using a 0.2 mm Ni–Cr thermocouple connected to a data logger
6
134
(THERM 3280-8M, Ahlborn Mess- und Regelungstechnik, Holzkirchen, Germany) and PC. An
135
illustrative temperature course is presented in Fig. 1. The experiments were carried out for each
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juice and inactivation temperature in triplicate. The heat transfer coefficient was estimated from a
137
simple dynamic enthalpy balance (Illeová, Polakovič, Štefuca, Ačai, & Juma, 2003):
138 139
dT K (TB T ) dt
(1a)
140
t=0
(1b)
T = 298.15 K
141 142
where T is the sample temperature, TB is the bath temperature, t is the heating time and K is the
143
proportionality factor including the overall heat transfer coefficient (Illeová et al., 2003). The
144
coefficient K was determined with a good accuracy and reproducibility when no effect of the
145
temperature dependence was found. The mean values of the coefficient used in further modelling
146
were 1.40 min-1 for broccoli juice, 1.63 min-1 for carrot juice and 1.68 min-1 for potato juice.
147 148
2.5. M o d e l l i n g
149 150
For each vegetable juice, all experimental data were modelled simultaneously using the
151
so-called multitemperature evaluation (Vrábel, Polakovič, Štefuca, & Báleš, 1997). A biphasic
152
isozyme mechanism (Sadana, 1991) was examined where the inactivation proceeds according to
153
the scheme:
154 155
k1 E1 I1
(2a)
7
156
k2 E2 I2
(2b)
157 158
where E1 and E2 are native isoforms, I1 and I2 are inactive forms, and k1 and k2 are the reaction
159
rate constants. The corresponding mathematical model consisted of ordinary differential
160
equations describing the changes of the concentrations of native isoforms, CE1 and CE2:
161
d CE1 k1CE1 dt
(3a)
162
d CE2 k2CE2 dt
(3b)
163 164
The second model was based on a combination of the isoenzyme inactivation mechanism
165
described above and the series mechanism of Lumry-Eyring (Lumry & Eyring, 1954) and is
166
represented by the following scheme:
167
k1 k3 D E1 I1
(4a)
168
k4 E2 I2
(4b)
k2
169 170
where D is a reversibly inactivated form. The model equations describing the concentration
171
changes of species are as follows:
172 173
d CE1 k1CE1 k2CD dt
(5a)
174
d CD k1CE1 k2CD k3CD dt
(5b)
175
d CE2 k4CE2 dt
(5c)
8
176 177
The concentrations in Eqs. (3a, b) and (5a–c) were substituted by relative activities, which
178
were obtained after the multiplication of the equations by the corresponding molar activities of
179
the enzymatic forms and the division by the total initial activity. An exception is the inactive
180
form D, which activity was formally obtained as a product of its concentration and molar activity
181
of E1. The initial conditions for both sets of differential equations were:
182 183
t0
aE1
aE2 1
aD 0
(6)
184 185
where aE1, aE2, and aD are the relative activities of the forms E1, E2, and D, respectively. The
186
fraction of the initial relative activity of isoform E1, α, was a fitted model parameter.
187 188
189
The temperature dependence of the kinetic rate constants of reactions was given by the Arrhenius equation:
ki ki 0e
Eai T0 1 RT0 T
i = 1–4
(7)
190 191
where ki are the individual rate constants, ki0 are the rate constants at the reference temperature of
192
T0 = 339.15 K, Eai are the corresponding activation energies and R = 8.314 J mol-1 K-1 is the gas
193
constant. Both models contained also the enthalpy balance (Eq. (1)).
194 195
All data fitting was performed using parameter estimation software Athena Visual Workbench 10.0 (Stewart & Associates Engineering Software, Madison, WI).
196 197
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198
3. Results and discuss ion
199 200
Thermal inactivation experiments of each vegetable POD were carried out at five different
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bath temperatures. They ranged from 62 ºC to 78 ºC for carrot and from 58 ºC to 74 ºC for other
202
three food materials. Higher temperatures for carrot juice were chosen because POD was more
203
stable in this material. No significant difference was observed between the inactivation rates of
204
POD in the two potato juices. The thermal stability of potato POD was thus not influenced by the
205
presence of 3 % onion. For that reason, only the inactivation of POD in simple potato mixture is
206
reported in this publication.
207
The results of all experiments are presented in Figs. 24. It is evident that the shapes of
208
the inactivation curves of individual POD's were noticeably different. Carrot and potato POD's
209
exhibited inactivation patterns typical for plant peroxidases which is characteristic by extremely
210
rapid inactivation in the first phase followed by several orders of magnitude slower rates in the
211
second phase (Khan & Robinson, 1993b; Forsyth et al., 1999; Lemos et al., 2000; Machado &
212
Saraiva, 2002). Both carrot and potato POD's lost more than 50 % of the initial activity during a
213
few minutes in the first phase. On the other hand, broccoli POD inactivation was biphasic with a
214
small deviation from first-order kinetics.
215
As has been mentioned above, the analysis of the kinetic data was based on the models
216
derived from mechanisms. Most publications on the inactivation of POD's found in fruits and
217
vegetables reported that these enzymes had many isoforms differing in thermal stability (Forsyth
218
et al., 1999; Johri et al., 2005; Khan & Robinson, 1993b; McLellan & Robinson, 1987; Rudra et
219
al., 2007; Yemenicioğlu et al., 1998). For that reason, the simple isozyme mechanism (Eq. (2))
220
was examined first. The model was formed by Eqs. (1a, b), (3a, b), and (6) and described the
10
221
inactivation of broccoli POD very well (Fig. 2). The mean square error of the relative activity was
222
2.32 % so the model could be considered adequate (Table 2). The kinetic parameters of the model
223
were estimated with a good accuracy too (Table 3). The initial fraction of the form E1 was 26 %.
224
The values of the rate constants at the reference temperature of 66 °C, k10 = 0.264 min-1 and k20 =
225
0.015 min-1, determined that the fraction 1 was the labile one and fraction 2 was the stable one.
226
These two fractions had a noticeable difference in the activation energies of inactivation which
227
were 71 kJ mol-1 and 333 kJ mol-1, respectively.
228
Table 2 further shows that mean square errors of the fits of the inactivation data of carrot
229
and potato POD's with the simple isozyme model were about 5 %. Significant deviations between
230
the experimental and model activity values were observed in the first phase of inactivation,
231
especially at lower temperatures (data not shown). The model was thus not adequate and a more
232
complex inactivation mechanism had to be considered. Following the existing knowledge
233
presented in the Introduction, an extended isozyme mechanism (Eq. (4)) was suggested for the
234
inactivation of carrot and potato POD's. The mechanism assumed that one of the isoforms
235
undergoes a biphasic inactivation according to the Lumry-Eyring mechanism whereas the second
236
one through a simple, irreversible one-step reaction. The model was formed by Eqs. (1a, b),
237
(5a-c), and (6).
238
The extension of the simple isozyme model resulted in a significant improvement of the
239
description of the inactivation of carrot and potato POD's. This is demonstrated in Figs. 3 and 4
240
by a good match of experimental and model data and in Table 2 by the reduction of the mean
241
square error of relative activity to 1.75 % and 2.21 %, respectively. The parameters of the models
242
and their uncertainties represented by the half-widths of 95% confidence intervals are presented
243
in Table 3. All parameters but the rate constants of the reversible reaction of the form E1 were
244
estimated equally well as for the inactivation of broccoli POD. The uncertainties of k10 and k20 11
245
were somewhat larger but still lower than the parameter values which makes the model credible.
246
The values of k10 and k20 were rather large what implies that the first-step had a character of rapid
247
equilibrium reaction. This step was responsible for the large drop of enzyme activity in the initial
248
phase but resulted in the formation of an intermediate form D which rate constant of the
249
transformation into an irreversibly inactivated form was much lower than that of the second
250
isozyme form E2. This is well illustrated in Fig. 5 on the courses of the relative activity of
251
individual peroxidase isoforms at the reference temperature of 66 C. The initial fractions of the
252
form E1 were 69 % for both carrot and potato POD, which is very close to the value of 74 % for
253
the stable fraction of broccoli POD.
254
The lowest activation energy, Ea, 70.7 kJ mol-1 was found for the inactivation of labile
255
isozyme of broccoli POD. Somewhat larger values, from 100.3 kJ mol-1 to 191.5 kJ mol-1, were
256
obtained for the activation energies of reversible reaction of carrot and potato POD's. The highest
257
activation energies, between 301 kJ mol-1 and 379 kJ mol-1, were estimated for the irreversible
258
reactions of potato, carrot and stable isoform of broccoli POD's. Unfortunately, is problematic to
259
compare these values to the values of activation energies presented in literature for simpler
260
mechanisms.
261 262 263
4. Concl usio ns
264 265
The investigation of the inactivation kinetics of broccoli, carrot and potato POD's revealed
266
the presence of two enzyme isoforms with distinct thermal stabilities in each vegetable material.
267
Labile and stable isozyme fractions were distributed in about the same proportion of 30:70 % in
268
all these materials but they differed in the inactivation kinetics. Whereas both broccoli isozymes 12
269
inactivated via first-order kinetics, the activity loss of the stable isoforms of potato and carrot
270
peroxidases was biphasic with a very fast, reversible transformation in the first step followed by a
271
slow, irreversible transformation of an intermediate. An interesting observation was that the
272
activation energies of the irreversible reactions of the stable forms were significantly larger than
273
those of reversible reactions or irreversible reaction of broccoli labile form.
274 275 276
Ac know ledgements
277
This study was supported by grants from the 6th Framework Program of EU, Project FOODPRO
278
(Ohmic heating for food processing), No. SME-2003-1-508374 and Slovak Grant Agency for
279
Science, VEGA 1/3582/06.
280 281 282
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351
Figure Ca ptions
352 353
Fig. 1. Heating profile of carrot juice at the bath temperature of 62 ºC. The symbols represent
354
experimental values and the solid line is a fitted course using Eq. (1).
355
Fig. 2. Thermal inactivation of broccoli POD. The symbols represent experimental data at the
356
temperatures of 58 C (◇), 62 C (■), 66 C (□), 70 °C (▲) and 74 C (△). The lines represent
357
a fit with the isozyme model. The inset depicts the initial phase of inactivation.
358
Fig. 3. Thermal inactivation of carrot POD. The symbols represent experimental data at the
359
temperatures of 62 C (■), 66 C (□), 70 °C (▲), 74 C (△) and 78 °C (◆). The lines represent
360
a fit with the combined isozyme and Lumry-Eyring model. The inset depicts the initial phase of
361
inactivation.
362
Fig. 4. Thermal inactivation of potato POD. The symbols represent experimental data at the
363
temperatures of 58 C (◇), 62 C (■), 66 C (□), 70 °C (▲) and 74 C (△). The lines represent
364
a fit with the combined isozyme and Lumry-Eyring model. The inset depicts the initial phase of
365
inactivation.
366
Fig. 5.Time course of activity loss of POD isoforms at 66 °C in different vegetable juices
367
evaluated from adequate models (Table 3). Broccoli POD – dashed lines, carrot POD – solid
368
lines, potato POD – dash-dotted lines.
369
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Figure 2 Click here to download high resolution image
Figure 3 Click here to download high resolution image
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Table 1
Table 1 Composition of vegetable mixtures Component
Broccoli
Carrot
Potato
Potato and onion
Vegetable [g]
370.0
370.00
369.40
360.00
Butter [g]
8.0
8.00
8.00
7.00
Milk [g]
1.40
1.40
2.00
2.00
Salt [g]
0.50
0.50
0.50
0.50
Pepper [g]
0.10
0.10
0.10
Spring onion [g]
10.50
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Table 2
Table 1 Mean square errors (MSE) and F-values (model variance divided by reproducibility variance) of POD activity data in vegetable juices for different models obtained by multi-temperature modelling. MSE [%]/F
Model Broccoli
Carrot
Potato
Isozyme
2.32/1.66
5.08/7.97
4.82/7.17
Combined isozyme & Lumry-Eyring
-
1.75/0.95
2.21/1.50
A model was considered adequate if its F-value was lower than the critical value of F for 60–69 vs. 15 degrees of freedom at the confidence level of 95% which was 2.15–2.16.
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Table 3
Table 1 Kinetic parameters of thermal inactivation of vegetable POD's obtained by the multitemperature evaluation of the data presented in Figs. 24 Isozyme model
Combined isozyme and Lumry-Eyring model
Broccoli POD
Carrot POD
Potato POD
k10 [min-1]
0.264 ± 0.057
3.48 ± 1.81
42.52 ± 25.17
k20 [min-1]
1.5010-2 ± 6.9210-4
3.78 ± 2.17
12.68 ± 8.08
k30 [min-1]
5.1910-3 ± 7.2710-4
6.4910-3 ± 1.6610-3
k40 [min-1]
0.254 ± 0.044
8.29 ± 3.74
0.264 ± 0.022
0.692 ± 0.025
0.689 ± 0.035
70.7 ± 34.2
100.3 ± 31.5
182.4 ± 30.1
104.3 ± 33.0
191.5 ± 33.5
Ea3 [kJ mol-1]
357.5 ± 20.8
301.2 ± 48.3
Ea4 [kJ mol-1]
329.6 ± 35.1
379.0 ± 68.2
Ea1 [kJ mol-1] Ea2 [kJ mol-1]
332.7 ± 6.1
The values after the plus/minus sign represent the half-widths of the 95% confidence intervals.
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