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Thermal inactivation kinetics of vegetable peroxidases
Journal of Food Engineering 91 (2009) 387–391
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Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Thermal inactivation kinetics of vegetable peroxidases Halina Połata, Alina Wilin´ska, Jolanta Bryjak, Milan Polakovicˇ * Faculty of Chemical and Food Technology, Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia
a r t i c l e
i n f o
Article history: Received 9 January 2008 Received in revised form 23 June 2008 Accepted 27 September 2008 Available online 4 October 2008 Keywords: Peroxidase Thermal processing Vegetable juice Enzyme stability Inactivation kinetics Multitemperature modelling
a b s t r a c t Thermal stability of peroxidases present in raw vegetable mixtures was investigated in order to identify adequate mechanisms and corresponding kinetic models of inactivation. Inactivation experiments were carried out for each material at five different temperatures which were from the ranges of 58–74 °C for broccoli and potato juices and 62–78 °C for carrot juice. Using the multitemperature evaluation of inactivation data, a simple isozyme model was verified for the inactivation of broccoli peroxidase. A combined three-reaction mechanism, which assumed simple irreversible inactivation for one isoform and Lumry–Eyring mechanism for the other one, was identified for carrot and potato peroxidases. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Consumers are interested in thermally processed food in which important nutritive compounds are damaged as little as possible. Therefore it is of great importance to specify proper conditions for food sterilization. For example, heat treatment of fruit and vegetable products should assure their microbiological safety, prevent browning and loss of colour, and simultaneously, it should not affect their natural qualities. To accomplish these requirements, a comprehensive study of thermal processing must be made. Peroxidases (PODs) are used as blanching indicators since they belong to the most stable and widespread plant enzymes and have a certain effect on the loss of colour and textural changes of fruits and vegetables (Yemeniciog˘lu et al., 1998; Forsyth et al., 1999; Icier et al., 2006). Their advantage compared to other potential blanching index enzymes is a simple, inexpensive activity measurement (Khan and Robinson, 1993b; Tijskens et al., 1997; Yemeniciog˘lu et al., 1998; Forsyth et al., 1999; Icier et al., 2006;). For example, horseradish peroxidase was used for the development of time–temperature integrators that are systems simulating temperature resistance of target microorganisms in thermal food processing (Lemos et al., 2000). POD is a monomeric, glycosylated protein containing haem as prosthetic group (McLellan and Robinson, 1987; Khan and Robinson, 1993a, 1993b; Yang et al., 1996; Tijskens et al., 1997; Forsyth and Robinson, 1998; Forsyth et al., 1999; Leon et al., 2002; Carv* Corresponding author. Tel.: +421 2 59325254; fax: +421 2 52496920. E-mail address: [email protected] (M. Polakovicˇ). 0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.09.017
alho et al., 2003; Wang et al., 2004; Johri et al., 2005). POD occurs in plant cells in both soluble and ionically bound isoforms that have different molecular masses, pI (Khan and Robinson, 1993a; Forsyth and Robinson, 1998; Johri et al., 2005), substrate specificity (Khan and Robinson, 1994) and thermal stability (McLellan and Robinson, 1987; Khan and Robinson, 1993b; Yemeniciog˘lu et al., 1998; Forsyth et al., 1999; Johri et al., 2005). The amount of covalently bounded carbohydrates significantly differs for POD isoforms or POD from different sources (Yang et al., 1996). The primary function of POD in plants is the reduction of hydrogen peroxide at the expense of oxidation of phenolic compounds. It is responsible for the mechanical properties of cell walls during extension, cell adhesion and disease resistance (Tijskens et al., 1997). The kinetics of POD catalytic action as well as its substrate specificity was widely studied (Khan and Robinson, 1993a, 1994; Forsyth and Robinson, 1998; Leon et al., 2002; Rani and Abraham, 2006; Kamal and Behere, 2003; Santos de Araujo et al., 2004; Johri et al., 2005). POD thermal inactivation was studied for purified isoperoxidases (Khan and Robinson, 1993b; Forsyth et al., 1999; Lemos et al., 2000; Machado and Saraiva, 2002; Carvalho et al., 2003; Kamal and Behere, 2003), in crude plant extracts (Khan and Robinson, 1993b; Yemeniciog˘lu et al., 1998; Quitão-Teixeira et al., 2008; Rudra et al., 2008) and in vegetable particles (Icier et al., 2006). Different inactivation mechanisms were identified. Khan and Robinson (1993b) found that even the inactivation mechanisms of highly purified isoforms are complex. They suggested a micro-heterogeneity of purified isozymes to be responsible for producing nonfirst-order inactivation plots. The origin of the heterogeneity was
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assigned to different moieties of covalently bound neutral carbohydrates. Machado and Saraiva (2002) found a biexponential model that described the inactivation kinetics of horseradish POD well but was not associated with any exact mechanism. An extensive study of thermal inactivation of an anionic horseradish POD was performed using differential scanning calorimetry, circular dichroism and tryptophan fluorescence (Carvalho et al., 2003). All three methods confirmed a hypothesis that the enzyme inactivation was a two-step process governed by the Lumry–Eyring mechanism. It was found that an inactive intermediate in distinction to a final, irreversibly denatured form was capable to incorporate a haem. Another type of series inactivation mechanism assuming partially inactivated intermediate species was postulated by Forsyth et al. (1999). Tijskens et al. (1997) who studied POD inactivation in slices of peaches, carrots and potatoes observed different behaviour of bound and soluble isoforms of the enzyme each characterized by first-order kinetics. Moreover, the bound form underwent a transition into the soluble form. Conventional heating was applied in this study in order to examine the inactivation of POD in broccoli, carrot and potato purees. The objective was to identify suitable mechanisms of POD inactivation and to obtain kinetic parameters that can be used in further analyses.
In order to determine the reproducibility error of the inactivation experiments, the whole experiment of inactivation of broccoli POD at 66 °C was duplicated and samples were taken in the same times. The variance of measured relative activity was first calculated for each time and the mean over all time values was then obtained. The square root of the mean variance, reproducibility error of relative activity, was 1.80 with 15 degrees of freedom. 2.3. Determination of POD activity
2. Materials and methods
The POD activity was determined at 25 °C. A sample with a volume of either 20 ll (carrot juice supernatant) or 70 ll (potato and broccoli juice supernatants) was added to 1.45 ml of 0.19 mM 2,2’azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS) in 100 mM Na-acetate buffer at pH 5.5. The reaction was initiated by adding 50 ll of 0.02% (carrot and broccoli juice supernatants) or 0.2 % (potato juice supernatant) H2O2. The sample volume and H2O2 concentration depended on the specific POD activity in different vegetables (Leon et al., 2002; Wang et al., 2004). The increase of absorbance at 405 nm (Wang et al., 2004) was recorded for 1–15 min using Cecil 9000 spectrophotometer (Cecil Instruments, Cambridge, UK). The activity was calculated from the slope of the absorbance vs. time dependence. The enzyme activity of 1 U corresponds to the rate of absorbance change of 0.001 min1 (Mdluli, 2005).
2.1. Vegetable juices
2.4. Heat transfer experiments
Four vegetable mixtures based on smashed carrot, broccoli, potatoes and potatoes with spring onion were prepared according to the recipes provided by Nature’s Best (Drogheda, Ireland). The mixtures contained about 97% of vegetable components. Their exact composition is given in Table 1. Vegetables, butter, salt and pepper were purchased in local shops and fresh milk was delivered by a dairy company (Rajo, Bratislava, Slovakia). Broccoli was washed and cut, whereas carrot and potatoes were first peeled. All ingredients of the mixtures were mixed, chopped with a blender and squeezed in a juice extractor. The juices obtained were portioned out into amounts needed for one experiment and frozen. The juices were defrosted at room temperature before their use in inactivation experiments.
Since the set inactivation temperature was reached in the entire sample only with a time delay, the analysis of sample thermal history was made and the values of the heat coefficients were estimated. Pre-incubated test tubes were filled with 0.8 ml of a sample and the temperature was recorded every 3 s using a 0.2 mm Ni–Cr thermocouple connected to a data logger (THERM 3280-8 M, Ahlborn Mess- und Regelungstechnik, Holzkirchen, Germany) and PC. An illustrative temperature course is presented in Fig. 1. The experiments were carried out for each juice and inactivation temperature in triplicate. The heat transfer coefficient was estimated from a simple dynamic enthalpy balance (Illeová et al., 2003):
2.2. Inactivation experiments
dT ¼ KðT B TÞ dt
ð1aÞ
t ¼ 0 T ¼ 298:15 K
ð1bÞ
Inactivation experiments were performed in 1.5 ml plastic test tubes that were pre-incubated in a water bath at an inactivation temperature and then filled with 0.8 ml of vegetable juice of ambient temperature. In specified time intervals, the tubes were taken out from the bath, immediately cooled down for 5 min in an ice– water/ethanol mixture (4 °C) and then kept in an ice–water bath until activity measurement. Potato and carrot juice samples were centrifuged at 12,000 for 15 min whereas broccoli juice samples were centrifuged at 14,000 rpm for 40 min. The supernatants were used for the determination of activity.
Table 1 Composition of vegetable mixtures. Component
Broccoli
Carrot
Potato
Potato and onion
Vegetable [g] Butter [g] Milk [g] Salt [g] Pepper [g] Spring onion [g]
370.0 8.0 1.40 0.50 0.10 –
370.00 8.00 1.40 0.50 0.10 –
369.40 8.00 2.00 0.50 0.10 –
360.00 7.00 2.00 0.50 – 10.50
Fig. 1. Heating profile of carrot juice at the bath temperature of 62 °C. The symbols represent experimental values and the solid line is a fitted course using Eq. (1).
H. Połata et al. / Journal of Food Engineering 91 (2009) 387–391
where T is the sample temperature, TB is the bath temperature, t is the heating time and K is the proportionality factor including the overall heat transfer coefficient (Illeová et al., 2003). The coefficient K was determined with a good accuracy and reproducibility when no effect of the temperature dependence was found. The mean values of the coefficient used in further modelling were 1.40 min1 for broccoli juice, 1.63 min1 for carrot juice and 1.68 min1 for potato juice.
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where ki are the individual rate constants, ki0 are the rate constants at the reference temperature of T0 = 339.15 K, Eai are the corresponding activation energies and R = 8.314 J mol1 K1 is the gas constant. Both models contained also the enthalpy balance Eq. (1). All data fitting was performed using parameter estimation software Athena Visual Workbench 10.0 (Stewart & Associates Engineering Software, Madison, WI). 3. Results and discussion
2.5. Modelling For each vegetable juice, all experimental data were modelled simultaneously using the so-called multitemperature evaluation (Vrábel et al., 1997). A biphasic isozyme mechanism (Sadana, 1991) was examined where the inactivation proceeds according to the scheme: k1
E1 ! I 1
ð2aÞ
k2
E2 ! I 2
ð2bÞ
where E1 and E2 are native isoforms, I1 and I2 are inactive forms, and k1 and k2 are the reaction rate constants. The corresponding mathematical model consisted of ordinary differential equations describing the changes of the concentrations of native isoforms, CE1 and CE2:
dC E1 ¼ k1 C E1 dt
ð3aÞ
dC E2 ¼ k2 C E2 dt
ð3bÞ
The second model was based on a combination of the isoenzyme inactivation mechanism described above and the series mechanism of Lumry–Eyring (Lumry and Eyring, 1954) and is represented by the following scheme: k1
k3
E1 ¢ D ! I 1
ð4aÞ
k2
k4
E2 ! I 2
ð4bÞ
where D is a reversibly inactivated form. The model equations describing the concentration changes of species are as follows:
dC E1 ¼ k1 C E1 þ k2 C D dt dC D ¼ k1 C E1 k2 C D k3 C D dt dC E2 ¼ k4 C E2 dt
ð5aÞ
Thermal inactivation experiments of each vegetable POD were carried out at five different bath temperatures. They ranged from 62 °C to 78 °C for carrot and from 58 °C to 74 °C for other three food materials. Higher temperatures for carrot juice were chosen because POD was more stable in this material. No significant difference was observed between the inactivation rates of POD in the two potato juices. The thermal stability of potato POD was thus not influenced by the presence of 3% onion. For that reason, only the inactivation of POD in simple potato mixture is reported in this publication. The results of all experiments are presented in Figs. 2–4. It is evident that the shapes of the inactivation curves of individual PODs were noticeably different. Carrot and potato PODs exhibited inactivation patterns typical for plant peroxidases which is characteristic by extremely rapid inactivation in the first phase followed by several orders of magnitude slower rates in the second phase (Khan and Robinson, 1993b; Forsyth et al., 1999; Lemos et al., 2000; Machado and Saraiva, 2002). Both carrot and potato PODs lost more than 50% of the initial activity during a few minutes in the first phase. On the other hand, broccoli POD inactivation was biphasic with a small deviation from first-order kinetics. As has been mentioned above, the analysis of the kinetic data was based on the models derived from mechanisms. Most publications on the inactivation of PODs found in fruits and vegetables reported that these enzymes had many isoforms differing in thermal stability (Forsyth et al., 1999; Johri et al., 2005; Khan and Robinson, 1993b; McLellan and Robinson, 1987; Rudra et al., 2008; Yemeniciog˘lu et al., 1998). For that reason, the simple isozyme mechanism (Eq. (2)) was examined first. The model was formed by Eq. (1a), (1b), (3b), (4b) and Eq. (6) and described the inactivation of broccoli POD very well (Fig. 2). The mean square error of the relative activity was 2.32% so the model could be considered adequate (Table 2). The kinetic parameters of the model were estimated with
ð5bÞ ð5cÞ
The concentrations in Eqs. (3a), (3b) and Eqs. (5a), (5b), (5c) were substituted by relative activities, which were obtained after the multiplication of the equations by the corresponding molar activities of the enzymatic forms and the division by the total initial activity. An exception is the inactive form D, which activity was formally obtained as a product of its concentration and molar activity of E1. The initial conditions for both sets of differential equations were:
t ¼ 0 aE1 ¼ a aE2 ¼ 1 a aD ¼ 0
ð6Þ
where aE1, aE2, and aD are the relative activities of the forms E1, E2, and D, respectively. The fraction of the initial relative activity of isoform E1, a, was a fitted model parameter.The temperature dependence of the kinetic rate constants of reactions was given by the Arrhenius equation:
ki ¼ ki0 e
Eai RT 0
T 1 T0
i¼14
ð7Þ
Fig. 2. Thermal inactivation of broccoli POD. The symbols represent experimental data at the temperatures of 58 °C (}), 62 °C (j), 66 °C (h), 70 °C (N) and 74 °C (4). The lines represent a fit with the isozyme model. The inset depicts the initial phase of inactivation.
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H. Połata et al. / Journal of Food Engineering 91 (2009) 387–391 Table 3 Kinetic parameters of thermal inactivation of vegetable PODs obtained by the multitemperature evaluation of the data presented in Figs. 2–4.
k10 [min1] k20 [min1] k30 [min1] k40 [min1]
a Ea1 Ea2 Ea3 Ea4
Fig. 3. Thermal inactivation of carrot POD. The symbols represent experimental data at the temperatures of 62 °C (j), 66 °C (h), 70 °C (N), 74 °C (4) and 78 °C (). The lines represent a fit with the combined isozyme and Lumry–Eyring model. The inset depicts the initial phase of inactivation.
Fig. 4. Thermal inactivation of potato POD. The symbols represent experimental data at the temperatures of 58 °C (}), 62 °C (j), 66 °C (h), 70 °C (N) and 74 °C (4). The lines represent a fit with the combined isozyme and Lumry–Eyring model. The inset depicts the initial phase of inactivation.
1
[kJ mol ] [kJ mol1] [kJ mol1] [kJ mol1]
Isozyme model
Combined isozyme and Lumry– Eyring model
Broccoli POD
Carrot POD
Potato POD
0.264 ± 0.057 1.50 102 ± 6.92 104 –
3.48 ± 1.81 3.78 ± 2.17
42.52 ± 25.17 12.68 ± 8.08
5.19 103 ± 7.27 104 0.254 ± 0.044 0.692 ± 0.025 100.3 ± 31.5 104.3 ± 33.0 357.5 ± 20.8 329.6 ± 35.1
6.49 103 ± 1.66 103 8.29 ± 3.74 0.689 ± 0.035 182.4 ± 30.1 191.5 ± 33.5 301.2 ± 48.3 379.0 ± 68.2
– 0.264 ± 0.022 70.7 ± 34.2 332.7 ± 6.1 – –
The values after the plus/minus sign represent the half-widths of the 95% confidence intervals.
Table 2 further shows that mean square errors of the fits of the inactivation data of carrot and potato PODs with the simple isozyme model were about 5%. Significant deviations between the experimental and model activity values were observed in the first phase of inactivation, especially at lower temperatures (data not shown). The model was thus not adequate and a more complex inactivation mechanism had to be considered. Following the existing knowledge presented in the Introduction, an extended isozyme mechanism (Eq. (4b)) was suggested for the inactivation of carrot and potato PODs. The mechanism assumed that one of the isoforms undergoes a biphasic inactivation according to the Lumry–Eyring mechanism whereas the second one through a simple, irreversible one-step reaction. The model was formed by Eqs. (1a), (1b), (5a) (5b) (5c) and (6). The extension of the simple isozyme model resulted in a significant improvement of the description of the inactivation of carrot and potato PODs. This is demonstrated in Figs. 3 and 4 by a good match of experimental and model data and in Table 2 by the reduction of the mean square error of relative activity to 1.75% and 2.21%, respectively. The parameters of the models and their uncertainties represented by the half-widths of 95% confidence intervals are presented in Table 3. All parameters but the rate constants of the reversible reaction of the form E1 were estimated equally well as for the inactivation of broccoli POD. The uncertainties of k10 and k20 were somewhat larger but still lower than the parameter values
a good accuracy too (Table 3). The initial fraction of the form E1 was 26%. The values of the rate constants at the reference temperature of 66 °C, k10 = 0.264 min1 and k20 = 0.015 min1, determined that the fraction 1 was the labile one and fraction 2 was the stable one. These two fractions had a noticeable difference in the activation energies of inactivation which were 71 kJ mol1 and 333 kJ mol1, respectively.
Table 2 Mean square errors and F-values (model variance divided by reproducibility variance) of POD activity data in vegetable juices for different models obtained by multitemperature modelling. Model
Isozyme Combined isozyme & Lumry–Eyring
MSE [%]/F Broccoli
Carrot
Potato
2.32/1.66 –
5.08/7.97 1.75/0.95
4.82/7.17 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.
Fig. 5. Time course of activity loss of POD isoforms at 66 °C in different vegetable juices evaluated from adequate models (Table 3). Broccoli POD – dashed lines, carrot POD – solid lines, potato POD – dash-dotted lines.
H. Połata et al. / Journal of Food Engineering 91 (2009) 387–391
which makes the model credible. The values of k10 and k20 were rather large what implies that the first-step had a character of rapid equilibrium reaction. This step was responsible for the large drop of enzyme activity in the initial phase but resulted in the formation of an intermediate form D which rate constant of the transformation into an irreversibly inactivated form was much lower than that of the second isozyme form E2. This is well illustrated in Fig. 5 on the courses of the relative activity of individual peroxidase isoforms at the reference temperature of 66 °C. The initial fractions of the form E1 were 69% for both carrot and potato POD, which is very close to the value of 74% for the stable fraction of broccoli POD. The lowest activation energy, Ea, 70.7 kJ mol1 was found for the inactivation of labile isozyme of broccoli POD. Somewhat larger values, from 100.3 kJ mol1 to 191.5 kJ mol1, were obtained for the activation energies of reversible reaction of carrot and potato PODs. The highest activation energies, between 301 kJ mol1 and 379 kJ mol1, were estimated for the irreversible reactions of potato, carrot and stable isoform of broccoli PODs. Unfortunately, it is problematic to compare these values to the values of activation energies presented in literature for simpler mechanisms. 4. Conclusions The investigation of the inactivation kinetics of broccoli, carrot and potato PODs revealed the presence of two enzyme isoforms with distinct thermal stabilities in each vegetable material. Labile and stable isozyme fractions were distributed in about the same proportion of 30:70% in all these materials but they differed in the inactivation kinetics. Whereas both broccoli isozymes inactivated via first-order kinetics, the activity loss of the stable isoforms of potato and carrot peroxidases was biphasic with a very fast, reversible transformation in the first step followed by a slow, irreversible transformation of an intermediate. An interesting observation was that the activation energies of the irreversible reactions of the stable forms were significantly larger than those of reversible reactions or irreversible reaction of broccoli labile form. Acknowledgements This study was supported by grants from the 6th Framework Program of EU, Project FOODPRO (Ohmic heating for food processing), No. SME-2003-1-508374 and Slovak Grant Agency for Science, VEGA 1/3582/06. References Carvalho, A.S.L., Melo, E.P.E., Ferreira, B.S., Neves-Petersen, M.T., Petersen, S.B., Aires-Barros, M.R., 2003. Heme and pH-dependent stability of an anionic horseradish peroxidase. Archives of Biochemistry and Biophysics 415 (2), 257– 267. Forsyth, J.L., Owusu Apenten, R.K., Robinson, D.S., 1999. The thermostability of purified isoperoxidases from Brassica oleracea VAR gemmifera. Food Chemistry 65 (1), 99–109.
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Forsyth, J.L., Robinson, D.S., 1998. Purification of Brussels sprout isoperoxidases. Food Chemistry 63 (2), 227–234. Icier, F., Yildiz, H., Baysal, T., 2006. Peroxidase inactivation and colour changes during ohmic blanching of pea puree. Journal of Food Engineering 74 (3), 424– 429. Illeová, V., Polakovicˇ, M., Štefuca, V., Acˇai, P., Juma, M., 2003. Experimental modelling of thermal inactivation of urease. Journal of Biotechnology 105 (3), 235–243. Johri, S., Jamwal, U., Rasool, S., Kumar, A., Verma, V., Qazi, G.N., 2005. Purification and characterization of peroxidases from Withania somnifera (AGB 002) and their ability to oxidize IAA. Plant Science 169 (6), 1014–1021. Kamal, J.K.A., Behere, D.V., 2003. Activity, stability and conformational flexibility of seed coat soybean peroxidase. Journal of Inorganic Biochemistry 94 (3), 236– 242. Khan, A.A., Robinson, D.S., 1993a. Purification of an anionic peroxidase isoenzyme from mango (Mangifera indica L. var chaunsa). Food Chemistry 46 (1), 61–64. Khan, A.A., Robinson, D.S., 1993b. The thermostability of purified mango isoperoxidases. Food Chemistry 47 (1), 53–59. Khan, A.A., Robinson, D.S., 1994. Hydrogen donor specificity of mango isoperoxidases. Food Chemistry 49 (4), 407–410. Lemos, M.A., Oliveira, J.C., Saraiva, J.A., 2000. Influence of pH on the thermal inactivation kinetics of horseradish peroxidase in aqueous solution. Lebensmittel-Wissenschaft und-Technologie-Food Science and Technology 33 (5), 362–368. Leon, J.C., Alpeeva, I.S., Chubar, T.A., Galaev, I.Y., Csoregi, E., Sakharov, I.Y., 2002. Purification and substrate specificity of peroxidase from sweet potato tubers. Plant Science 163 (5), 1011–1019. Lumry, R., Eyring, H., 1954. Conformation changes of proteins. Journal of Physical Chemistry 58 (1), 110–120. Machado, M.F., Saraiva, J., 2002. Inactivation and reactivation kinetics of horseradish peroxidase in phosphate buffer and buffer-dimethylformamide solutions. Journal of Molecular Catalysis B: Enzymatic, 451–457. McLellan, K.M., Robinson, D.S., 1987. Purification and heat-stability of Brusselssprout peroxidase isoenzymes. Food Chemistry 23 (4), 305–319. Mdluli, K.M., 2005. Partial purification and characterisation of polyphenol oxidase and peroxidase from marula fruit (Sclerocarya birrea subsp. Caffra). Food Chemistry 92 (2), 311–323. Quitão-Teixeira, L.J., Aguiló-Aguayo, I., Ramos, A.M., Martín-Belloso, O., 2008. Inactivation of oxidative enzymes by high-intensity pulsed electric field for retention of color in carrot juice. Food and Bioprocess Technology. 1 (4), 364– 373. Rani, D.N., Abraham, T.E., 2006. Kinetic study of a purified anionic peroxidase isolated from Eupatorium odoratum and its novel application as time temperature indicator for food materials. Journal of Food Engineering 77 (3), 594–600. Rudra, S.G., Shivhare, U.S., Basu, S., Sarkar, B.C., 2008. Thermal inactivation kinetics of peroxidase in coriander leaves. Food and Bioprocess Technology. 1 (2), 187– 195. Sadana, A., 1991. Biocatalysis. Fundamentals of Enzyme Deactivation Kinetics. Prentice Hall, Englewood Cliffs, New Jersey. Santos de Araujo, B., Omena de Oliveira, J., Salgueiro Machado, S., Pletsch, M., 2004. Comparative studies of the peroxidases from hairy roots of Daucus carota, Ipomoea batatas and Solanum aviculare. Plant Science 167 (5), 1151–1157. Tijskens, L.M.M., Rodis, P.S., Hertog, M.L.A.T.M., Waldron, K.W., Ingham, L., Proxenia, N., van Dijk, C., 1997. Activity of peroxidase during blanching of peaches, carrots and potatoes. Journal of Food Engineering 34 (4), 355–370. Vrábel, P., Polakovicˇ, M., Štefuca, V., Báleš, V., 1997. Analysis of mechanism and kinetics of thermal inactivation of enzymes: evaluation of multitemperature data applied to inactivation of yeast invertase. Enzyme and Microbial Technology 20 (5), 348–354. Wang, L., Burhenne, K., Kristensen, B.K., Rasmussen, S.K., 2004. Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in Brassicaceae. Gene 343 (2), 323–335. Yang, B.Y., Gray, J.S.S., Montgomery, R., 1996. The glycans of horseradish peroxidase. Carbohydrate Research 287 (2), 203–212. Yemeniciog˘lu, A., Özkan, M., Veliog˘lu, S., & Cemerog˘lu, B., 1998. Thermal inactivation kinetics of peroxidase and lipoxygenase from fresh pinto beans (Phaseolus vulgaris). Zeitschrift für Lebensmitteluntersuchung und -Forschung A, 206(4), 294–296.
Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: www.elsevier.com/locate/jfoodeng
Thermal inactivation kinetics of vegetable peroxidases Halina Połata, Alina Wilin´ska, Jolanta Bryjak, Milan Polakovicˇ * Faculty of Chemical and Food Technology, Department of Chemical and Biochemical Engineering, Institute of Chemical and Environmental Engineering, Slovak University of Technology, Radlinského 9, 812 37 Bratislava, Slovakia
a r t i c l e
i n f o
Article history: Received 9 January 2008 Received in revised form 23 June 2008 Accepted 27 September 2008 Available online 4 October 2008 Keywords: Peroxidase Thermal processing Vegetable juice Enzyme stability Inactivation kinetics Multitemperature modelling
a b s t r a c t Thermal stability of peroxidases present in raw vegetable mixtures was investigated in order to identify adequate mechanisms and corresponding kinetic models of inactivation. Inactivation experiments were carried out for each material at five different temperatures which were from the ranges of 58–74 °C for broccoli and potato juices and 62–78 °C for carrot juice. Using the multitemperature evaluation of inactivation data, a simple isozyme model was verified for the inactivation of broccoli peroxidase. A combined three-reaction mechanism, which assumed simple irreversible inactivation for one isoform and Lumry–Eyring mechanism for the other one, was identified for carrot and potato peroxidases. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Consumers are interested in thermally processed food in which important nutritive compounds are damaged as little as possible. Therefore it is of great importance to specify proper conditions for food sterilization. For example, heat treatment of fruit and vegetable products should assure their microbiological safety, prevent browning and loss of colour, and simultaneously, it should not affect their natural qualities. To accomplish these requirements, a comprehensive study of thermal processing must be made. Peroxidases (PODs) are used as blanching indicators since they belong to the most stable and widespread plant enzymes and have a certain effect on the loss of colour and textural changes of fruits and vegetables (Yemeniciog˘lu et al., 1998; Forsyth et al., 1999; Icier et al., 2006). Their advantage compared to other potential blanching index enzymes is a simple, inexpensive activity measurement (Khan and Robinson, 1993b; Tijskens et al., 1997; Yemeniciog˘lu et al., 1998; Forsyth et al., 1999; Icier et al., 2006;). For example, horseradish peroxidase was used for the development of time–temperature integrators that are systems simulating temperature resistance of target microorganisms in thermal food processing (Lemos et al., 2000). POD is a monomeric, glycosylated protein containing haem as prosthetic group (McLellan and Robinson, 1987; Khan and Robinson, 1993a, 1993b; Yang et al., 1996; Tijskens et al., 1997; Forsyth and Robinson, 1998; Forsyth et al., 1999; Leon et al., 2002; Carv* Corresponding author. Tel.: +421 2 59325254; fax: +421 2 52496920. E-mail address: [email protected] (M. Polakovicˇ). 0260-8774/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2008.09.017
alho et al., 2003; Wang et al., 2004; Johri et al., 2005). POD occurs in plant cells in both soluble and ionically bound isoforms that have different molecular masses, pI (Khan and Robinson, 1993a; Forsyth and Robinson, 1998; Johri et al., 2005), substrate specificity (Khan and Robinson, 1994) and thermal stability (McLellan and Robinson, 1987; Khan and Robinson, 1993b; Yemeniciog˘lu et al., 1998; Forsyth et al., 1999; Johri et al., 2005). The amount of covalently bounded carbohydrates significantly differs for POD isoforms or POD from different sources (Yang et al., 1996). The primary function of POD in plants is the reduction of hydrogen peroxide at the expense of oxidation of phenolic compounds. It is responsible for the mechanical properties of cell walls during extension, cell adhesion and disease resistance (Tijskens et al., 1997). The kinetics of POD catalytic action as well as its substrate specificity was widely studied (Khan and Robinson, 1993a, 1994; Forsyth and Robinson, 1998; Leon et al., 2002; Rani and Abraham, 2006; Kamal and Behere, 2003; Santos de Araujo et al., 2004; Johri et al., 2005). POD thermal inactivation was studied for purified isoperoxidases (Khan and Robinson, 1993b; Forsyth et al., 1999; Lemos et al., 2000; Machado and Saraiva, 2002; Carvalho et al., 2003; Kamal and Behere, 2003), in crude plant extracts (Khan and Robinson, 1993b; Yemeniciog˘lu et al., 1998; Quitão-Teixeira et al., 2008; Rudra et al., 2008) and in vegetable particles (Icier et al., 2006). Different inactivation mechanisms were identified. Khan and Robinson (1993b) found that even the inactivation mechanisms of highly purified isoforms are complex. They suggested a micro-heterogeneity of purified isozymes to be responsible for producing nonfirst-order inactivation plots. The origin of the heterogeneity was
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assigned to different moieties of covalently bound neutral carbohydrates. Machado and Saraiva (2002) found a biexponential model that described the inactivation kinetics of horseradish POD well but was not associated with any exact mechanism. An extensive study of thermal inactivation of an anionic horseradish POD was performed using differential scanning calorimetry, circular dichroism and tryptophan fluorescence (Carvalho et al., 2003). All three methods confirmed a hypothesis that the enzyme inactivation was a two-step process governed by the Lumry–Eyring mechanism. It was found that an inactive intermediate in distinction to a final, irreversibly denatured form was capable to incorporate a haem. Another type of series inactivation mechanism assuming partially inactivated intermediate species was postulated by Forsyth et al. (1999). Tijskens et al. (1997) who studied POD inactivation in slices of peaches, carrots and potatoes observed different behaviour of bound and soluble isoforms of the enzyme each characterized by first-order kinetics. Moreover, the bound form underwent a transition into the soluble form. Conventional heating was applied in this study in order to examine the inactivation of POD in broccoli, carrot and potato purees. The objective was to identify suitable mechanisms of POD inactivation and to obtain kinetic parameters that can be used in further analyses.
In order to determine the reproducibility error of the inactivation experiments, the whole experiment of inactivation of broccoli POD at 66 °C was duplicated and samples were taken in the same times. The variance of measured relative activity was first calculated for each time and the mean over all time values was then obtained. The square root of the mean variance, reproducibility error of relative activity, was 1.80 with 15 degrees of freedom. 2.3. Determination of POD activity
2. Materials and methods
The POD activity was determined at 25 °C. A sample with a volume of either 20 ll (carrot juice supernatant) or 70 ll (potato and broccoli juice supernatants) was added to 1.45 ml of 0.19 mM 2,2’azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)diammonium salt (ABTS) in 100 mM Na-acetate buffer at pH 5.5. The reaction was initiated by adding 50 ll of 0.02% (carrot and broccoli juice supernatants) or 0.2 % (potato juice supernatant) H2O2. The sample volume and H2O2 concentration depended on the specific POD activity in different vegetables (Leon et al., 2002; Wang et al., 2004). The increase of absorbance at 405 nm (Wang et al., 2004) was recorded for 1–15 min using Cecil 9000 spectrophotometer (Cecil Instruments, Cambridge, UK). The activity was calculated from the slope of the absorbance vs. time dependence. The enzyme activity of 1 U corresponds to the rate of absorbance change of 0.001 min1 (Mdluli, 2005).
2.1. Vegetable juices
2.4. Heat transfer experiments
Four vegetable mixtures based on smashed carrot, broccoli, potatoes and potatoes with spring onion were prepared according to the recipes provided by Nature’s Best (Drogheda, Ireland). The mixtures contained about 97% of vegetable components. Their exact composition is given in Table 1. Vegetables, butter, salt and pepper were purchased in local shops and fresh milk was delivered by a dairy company (Rajo, Bratislava, Slovakia). Broccoli was washed and cut, whereas carrot and potatoes were first peeled. All ingredients of the mixtures were mixed, chopped with a blender and squeezed in a juice extractor. The juices obtained were portioned out into amounts needed for one experiment and frozen. The juices were defrosted at room temperature before their use in inactivation experiments.
Since the set inactivation temperature was reached in the entire sample only with a time delay, the analysis of sample thermal history was made and the values of the heat coefficients were estimated. Pre-incubated test tubes were filled with 0.8 ml of a sample and the temperature was recorded every 3 s using a 0.2 mm Ni–Cr thermocouple connected to a data logger (THERM 3280-8 M, Ahlborn Mess- und Regelungstechnik, Holzkirchen, Germany) and PC. An illustrative temperature course is presented in Fig. 1. The experiments were carried out for each juice and inactivation temperature in triplicate. The heat transfer coefficient was estimated from a simple dynamic enthalpy balance (Illeová et al., 2003):
2.2. Inactivation experiments
dT ¼ KðT B TÞ dt
ð1aÞ
t ¼ 0 T ¼ 298:15 K
ð1bÞ
Inactivation experiments were performed in 1.5 ml plastic test tubes that were pre-incubated in a water bath at an inactivation temperature and then filled with 0.8 ml of vegetable juice of ambient temperature. In specified time intervals, the tubes were taken out from the bath, immediately cooled down for 5 min in an ice– water/ethanol mixture (4 °C) and then kept in an ice–water bath until activity measurement. Potato and carrot juice samples were centrifuged at 12,000 for 15 min whereas broccoli juice samples were centrifuged at 14,000 rpm for 40 min. The supernatants were used for the determination of activity.
Table 1 Composition of vegetable mixtures. Component
Broccoli
Carrot
Potato
Potato and onion
Vegetable [g] Butter [g] Milk [g] Salt [g] Pepper [g] Spring onion [g]
370.0 8.0 1.40 0.50 0.10 –
370.00 8.00 1.40 0.50 0.10 –
369.40 8.00 2.00 0.50 0.10 –
360.00 7.00 2.00 0.50 – 10.50
Fig. 1. Heating profile of carrot juice at the bath temperature of 62 °C. The symbols represent experimental values and the solid line is a fitted course using Eq. (1).
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where T is the sample temperature, TB is the bath temperature, t is the heating time and K is the proportionality factor including the overall heat transfer coefficient (Illeová et al., 2003). The coefficient K was determined with a good accuracy and reproducibility when no effect of the temperature dependence was found. The mean values of the coefficient used in further modelling were 1.40 min1 for broccoli juice, 1.63 min1 for carrot juice and 1.68 min1 for potato juice.
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where ki are the individual rate constants, ki0 are the rate constants at the reference temperature of T0 = 339.15 K, Eai are the corresponding activation energies and R = 8.314 J mol1 K1 is the gas constant. Both models contained also the enthalpy balance Eq. (1). All data fitting was performed using parameter estimation software Athena Visual Workbench 10.0 (Stewart & Associates Engineering Software, Madison, WI). 3. Results and discussion
2.5. Modelling For each vegetable juice, all experimental data were modelled simultaneously using the so-called multitemperature evaluation (Vrábel et al., 1997). A biphasic isozyme mechanism (Sadana, 1991) was examined where the inactivation proceeds according to the scheme: k1
E1 ! I 1
ð2aÞ
k2
E2 ! I 2
ð2bÞ
where E1 and E2 are native isoforms, I1 and I2 are inactive forms, and k1 and k2 are the reaction rate constants. The corresponding mathematical model consisted of ordinary differential equations describing the changes of the concentrations of native isoforms, CE1 and CE2:
dC E1 ¼ k1 C E1 dt
ð3aÞ
dC E2 ¼ k2 C E2 dt
ð3bÞ
The second model was based on a combination of the isoenzyme inactivation mechanism described above and the series mechanism of Lumry–Eyring (Lumry and Eyring, 1954) and is represented by the following scheme: k1
k3
E1 ¢ D ! I 1
ð4aÞ
k2
k4
E2 ! I 2
ð4bÞ
where D is a reversibly inactivated form. The model equations describing the concentration changes of species are as follows:
dC E1 ¼ k1 C E1 þ k2 C D dt dC D ¼ k1 C E1 k2 C D k3 C D dt dC E2 ¼ k4 C E2 dt
ð5aÞ
Thermal inactivation experiments of each vegetable POD were carried out at five different bath temperatures. They ranged from 62 °C to 78 °C for carrot and from 58 °C to 74 °C for other three food materials. Higher temperatures for carrot juice were chosen because POD was more stable in this material. No significant difference was observed between the inactivation rates of POD in the two potato juices. The thermal stability of potato POD was thus not influenced by the presence of 3% onion. For that reason, only the inactivation of POD in simple potato mixture is reported in this publication. The results of all experiments are presented in Figs. 2–4. It is evident that the shapes of the inactivation curves of individual PODs were noticeably different. Carrot and potato PODs exhibited inactivation patterns typical for plant peroxidases which is characteristic by extremely rapid inactivation in the first phase followed by several orders of magnitude slower rates in the second phase (Khan and Robinson, 1993b; Forsyth et al., 1999; Lemos et al., 2000; Machado and Saraiva, 2002). Both carrot and potato PODs lost more than 50% of the initial activity during a few minutes in the first phase. On the other hand, broccoli POD inactivation was biphasic with a small deviation from first-order kinetics. As has been mentioned above, the analysis of the kinetic data was based on the models derived from mechanisms. Most publications on the inactivation of PODs found in fruits and vegetables reported that these enzymes had many isoforms differing in thermal stability (Forsyth et al., 1999; Johri et al., 2005; Khan and Robinson, 1993b; McLellan and Robinson, 1987; Rudra et al., 2008; Yemeniciog˘lu et al., 1998). For that reason, the simple isozyme mechanism (Eq. (2)) was examined first. The model was formed by Eq. (1a), (1b), (3b), (4b) and Eq. (6) and described the inactivation of broccoli POD very well (Fig. 2). The mean square error of the relative activity was 2.32% so the model could be considered adequate (Table 2). The kinetic parameters of the model were estimated with
ð5bÞ ð5cÞ
The concentrations in Eqs. (3a), (3b) and Eqs. (5a), (5b), (5c) were substituted by relative activities, which were obtained after the multiplication of the equations by the corresponding molar activities of the enzymatic forms and the division by the total initial activity. An exception is the inactive form D, which activity was formally obtained as a product of its concentration and molar activity of E1. The initial conditions for both sets of differential equations were:
t ¼ 0 aE1 ¼ a aE2 ¼ 1 a aD ¼ 0
ð6Þ
where aE1, aE2, and aD are the relative activities of the forms E1, E2, and D, respectively. The fraction of the initial relative activity of isoform E1, a, was a fitted model parameter.The temperature dependence of the kinetic rate constants of reactions was given by the Arrhenius equation:
ki ¼ ki0 e
Eai RT 0
T 1 T0
i¼14
ð7Þ
Fig. 2. Thermal inactivation of broccoli POD. The symbols represent experimental data at the temperatures of 58 °C (}), 62 °C (j), 66 °C (h), 70 °C (N) and 74 °C (4). The lines represent a fit with the isozyme model. The inset depicts the initial phase of inactivation.
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H. Połata et al. / Journal of Food Engineering 91 (2009) 387–391 Table 3 Kinetic parameters of thermal inactivation of vegetable PODs obtained by the multitemperature evaluation of the data presented in Figs. 2–4.
k10 [min1] k20 [min1] k30 [min1] k40 [min1]
a Ea1 Ea2 Ea3 Ea4
Fig. 3. Thermal inactivation of carrot POD. The symbols represent experimental data at the temperatures of 62 °C (j), 66 °C (h), 70 °C (N), 74 °C (4) and 78 °C (). The lines represent a fit with the combined isozyme and Lumry–Eyring model. The inset depicts the initial phase of inactivation.
Fig. 4. Thermal inactivation of potato POD. The symbols represent experimental data at the temperatures of 58 °C (}), 62 °C (j), 66 °C (h), 70 °C (N) and 74 °C (4). The lines represent a fit with the combined isozyme and Lumry–Eyring model. The inset depicts the initial phase of inactivation.
1
[kJ mol ] [kJ mol1] [kJ mol1] [kJ mol1]
Isozyme model
Combined isozyme and Lumry– Eyring model
Broccoli POD
Carrot POD
Potato POD
0.264 ± 0.057 1.50 102 ± 6.92 104 –
3.48 ± 1.81 3.78 ± 2.17
42.52 ± 25.17 12.68 ± 8.08
5.19 103 ± 7.27 104 0.254 ± 0.044 0.692 ± 0.025 100.3 ± 31.5 104.3 ± 33.0 357.5 ± 20.8 329.6 ± 35.1
6.49 103 ± 1.66 103 8.29 ± 3.74 0.689 ± 0.035 182.4 ± 30.1 191.5 ± 33.5 301.2 ± 48.3 379.0 ± 68.2
– 0.264 ± 0.022 70.7 ± 34.2 332.7 ± 6.1 – –
The values after the plus/minus sign represent the half-widths of the 95% confidence intervals.
Table 2 further shows that mean square errors of the fits of the inactivation data of carrot and potato PODs with the simple isozyme model were about 5%. Significant deviations between the experimental and model activity values were observed in the first phase of inactivation, especially at lower temperatures (data not shown). The model was thus not adequate and a more complex inactivation mechanism had to be considered. Following the existing knowledge presented in the Introduction, an extended isozyme mechanism (Eq. (4b)) was suggested for the inactivation of carrot and potato PODs. The mechanism assumed that one of the isoforms undergoes a biphasic inactivation according to the Lumry–Eyring mechanism whereas the second one through a simple, irreversible one-step reaction. The model was formed by Eqs. (1a), (1b), (5a) (5b) (5c) and (6). The extension of the simple isozyme model resulted in a significant improvement of the description of the inactivation of carrot and potato PODs. This is demonstrated in Figs. 3 and 4 by a good match of experimental and model data and in Table 2 by the reduction of the mean square error of relative activity to 1.75% and 2.21%, respectively. The parameters of the models and their uncertainties represented by the half-widths of 95% confidence intervals are presented in Table 3. All parameters but the rate constants of the reversible reaction of the form E1 were estimated equally well as for the inactivation of broccoli POD. The uncertainties of k10 and k20 were somewhat larger but still lower than the parameter values
a good accuracy too (Table 3). The initial fraction of the form E1 was 26%. The values of the rate constants at the reference temperature of 66 °C, k10 = 0.264 min1 and k20 = 0.015 min1, determined that the fraction 1 was the labile one and fraction 2 was the stable one. These two fractions had a noticeable difference in the activation energies of inactivation which were 71 kJ mol1 and 333 kJ mol1, respectively.
Table 2 Mean square errors and F-values (model variance divided by reproducibility variance) of POD activity data in vegetable juices for different models obtained by multitemperature modelling. Model
Isozyme Combined isozyme & Lumry–Eyring
MSE [%]/F Broccoli
Carrot
Potato
2.32/1.66 –
5.08/7.97 1.75/0.95
4.82/7.17 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.
Fig. 5. Time course of activity loss of POD isoforms at 66 °C in different vegetable juices evaluated from adequate models (Table 3). Broccoli POD – dashed lines, carrot POD – solid lines, potato POD – dash-dotted lines.
H. Połata et al. / Journal of Food Engineering 91 (2009) 387–391
which makes the model credible. The values of k10 and k20 were rather large what implies that the first-step had a character of rapid equilibrium reaction. This step was responsible for the large drop of enzyme activity in the initial phase but resulted in the formation of an intermediate form D which rate constant of the transformation into an irreversibly inactivated form was much lower than that of the second isozyme form E2. This is well illustrated in Fig. 5 on the courses of the relative activity of individual peroxidase isoforms at the reference temperature of 66 °C. The initial fractions of the form E1 were 69% for both carrot and potato POD, which is very close to the value of 74% for the stable fraction of broccoli POD. The lowest activation energy, Ea, 70.7 kJ mol1 was found for the inactivation of labile isozyme of broccoli POD. Somewhat larger values, from 100.3 kJ mol1 to 191.5 kJ mol1, were obtained for the activation energies of reversible reaction of carrot and potato PODs. The highest activation energies, between 301 kJ mol1 and 379 kJ mol1, were estimated for the irreversible reactions of potato, carrot and stable isoform of broccoli PODs. Unfortunately, it is problematic to compare these values to the values of activation energies presented in literature for simpler mechanisms. 4. Conclusions The investigation of the inactivation kinetics of broccoli, carrot and potato PODs revealed the presence of two enzyme isoforms with distinct thermal stabilities in each vegetable material. Labile and stable isozyme fractions were distributed in about the same proportion of 30:70% in all these materials but they differed in the inactivation kinetics. Whereas both broccoli isozymes inactivated via first-order kinetics, the activity loss of the stable isoforms of potato and carrot peroxidases was biphasic with a very fast, reversible transformation in the first step followed by a slow, irreversible transformation of an intermediate. An interesting observation was that the activation energies of the irreversible reactions of the stable forms were significantly larger than those of reversible reactions or irreversible reaction of broccoli labile form. Acknowledgements This study was supported by grants from the 6th Framework Program of EU, Project FOODPRO (Ohmic heating for food processing), No. SME-2003-1-508374 and Slovak Grant Agency for Science, VEGA 1/3582/06. References Carvalho, A.S.L., Melo, E.P.E., Ferreira, B.S., Neves-Petersen, M.T., Petersen, S.B., Aires-Barros, M.R., 2003. Heme and pH-dependent stability of an anionic horseradish peroxidase. Archives of Biochemistry and Biophysics 415 (2), 257– 267. Forsyth, J.L., Owusu Apenten, R.K., Robinson, D.S., 1999. The thermostability of purified isoperoxidases from Brassica oleracea VAR gemmifera. Food Chemistry 65 (1), 99–109.
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