Kinetics Of Peroxidase Inactivation Biology Essay

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Thermal treatment of seedless guava was done in the temperature range of 80-95°C.The kinetics of peroxidase inactivation and colour changes due to thermal treatments were determined. The thermodynamic (activation energy) and kinetic (rate constant and decimal reduction time) parameters for peroxidase inactivation and colour parameters changes were calculated. Peroxidase inactivation kinetics followed a first order kinetic model, where the activation energy was 96.39±4 kJ mol-1. Colour was quantified using the L, a, b system. The color changes during processing were described by a first order kinetic model except total colour difference which followed a zero order kinetic model. The temperature dependence of the degradation followed the Arrhenius relation. The activation energies (Ea) for L, a, b and total colour difference (TCD) were calculated and were 122.68±3, 88.47±5, 104.86±5 kJmol-1 and 112.65±5 kJmol-1, respectively. The results of this work are a good tool to further optimize seedless guava thermal treatment conditions.

Keywords: Blanching; Colour changes; Kinetic parameter; Peroxidase inactivation;

Seedless Guava.


Guava, Psidium guajava L., is native to the Caribbean and common throughout all warm areas of tropical America and in the West Indies. The fruits are oblong to pear shape, its skin is thin, and the flesh is white, red or salmon-colored. The fruit has many seeds in the centre though seedless varieties are also available. In Malaysia commercial guava production began in the mid 1980s and consumed as a fruit, has great amount of vitamin C (more than 3 times as much Vitamin C as an orange), vitamins A and B. Guavas are useful sources of nicotinic acid, phosphorous, and soluble fiber. Guava's are cholesterol and sodium free, plus low in fat and calories (Morton 1978).

Thermal processing is one of the most utilized methods for stabilising foods primarily intended to inactivate enzymes and destroy microorganisms. However, during processing, the food material may be exposed to temperatures that have an adverse effect on quality (loss of texture and unwanted changes of colour) and reduce the contents or bioavailability of some nutrients (Barreiro et al. 1997; Avila and Silva, 1999; Ibarz et al. 1999).

Generally, peroxidase is recognized as being one of the most heat-stable enzymes in fruits and vegetables and considered to be the indicator enzyme for adequacy of thermal process. Consequently, the absence of residual activity of this enzyme would indicate that other

less-resistant enzymes are also destroyed (Williams et al. 1986). The presence of residual peroxidase in processed products may cause quality changes, such as texture, colour, flavor and nutritional losses, however its role on quality impact during food storage is not clear yet. For these reasons, it is desirable to keep blanching treatment conditions at a level strictly sufficient to cause inactivation of the deleterious enzymes, to minimize quality losses.

The first quality judgment made by a consumer on a food at the point of sale is its visual appearance. Colour is one of the most important appearance attribute of food materials, since it influences consumer acceptability. Furthermore, colour of a processed product is often expected to be as similar as possible to the raw one (MacDougall 2002). Therefore, maintaining the natural colour in processed fruits and vegetables has been a major challenge in food processing. Changes in fruits and vegetables colour can be associated with its previous heat treatment history and is also an indicator of heat treatment severity. Various reactions such as pigment destruction (carotenoids and chlorophylls) and non-enzymatic browning (Maillard condensation of hexoses and amino components) reactions can occur during thermal processing of fruits and vegetables and therefore affect its colour (Reyes and Luh 1960; Abets and Wrolstad 1979; Resnik and Chirife 1979; Cornwell and Wrolstad 1981; Cinar 2004). The retention of total colour can be used as a quality indicator to evaluate the extent of deterioration due to thermal processing (Shin and Bhowmik 1995).

Knowledge on degradation kinetics of enzyme inactivation and quality changes including the reaction order, the reaction constant, and the energy of activation is essential to predict quality losses during thermal processes. Several researchers have published work on enzyme inactivation and thermal degradation kinetics of colour in different range of temperature. The majority of the published work report, enzyme inactivation and colour changes are well described by zero, first-order models (Anthon and Barrett 2002; Soysal and Soylemez 2005; Aguerre and Suarez 1987; Rhim et al. 1989; Ahmed et al. 2002; Baik and Mittal 2003; Barreiro et al. 1997; Ibarz et al. 1999; Nisha et al. 2004; Rattanathanalerk et al. 2005) or the fractional conversion (also known as reversible first order model), (Ávila and Silva 1999; Steet and Tong 1996; Cruz et al. 2007). There is currently no published data for the kinetics of thermal inactivation of peroxidase and colour changes in seedless guava. We report here more detailed kinetic data for thermal inactivation of peroxidase and colour changes during thermal treatments. Therefore, the aim of this study was to determine the kinetic parameters for peroxidase inactivation and colour changes of seedless guava. This information will help to optimize thermal process for seedless guava.



Raw seedless guavas (Psidium guajava L.) at commercial maturity were purchased from a local market in Serdang, Malaysia. The fruits were washed and peeled. Then, they were cut into cubes (2cmÃ-2cmÃ-2cm). All chemicals used in this study were analytical grade.

Thermal Treatment

Seedless guavas (Psidium guajava L.) were blanched in a circulating water bath (Memmert, WNE14. Memmert GmbH Co. KG, Germany) maintained at desired temperatures (±0.5°C). Heat inactivation was studied at temperatures ranging from 80 to 95ËšC, with different times of exposure. After preset times, the samples were removed from the water bath and placed immediately in cooled water (2-5ËšC) in order to stop thermal inactivation instantaneously. The temperature of the water bath and cooled water was verified with a digital thermometer (Ellab CTD-85, Ellab, Denmark) and a thermocouple (1.2 mm needle diameter constantan-type T). Each experiment was replicated trice. An unblanched sample was taken as control.

Enzyme Extraction Procedure

In order to determine the presence of peroxidase in seedless guava and ratio between sample weight (g) and the buffer solution volume (mL), preliminary experiments were carried out. Blanched samples were mixed with cold potassium phosphate buffer in the proportion of 3:25 w/v. Each sample was homogenized in an Ultra-Turrax T25 Janke & Kunkel for 1 min at 13,500 rpm under chilled condition. The homogenate was filtered using filter paper (Whatman No.1). The filtrate was centrifuged in a Beckman Coulter, Avanti J-25 centrifuge with a rotor no.JA14 at 6000 Ã- g and 4ËšC for 20 min with polypropylene tubes. The supernatants were kept on ice until the analysis.

Determination of Peroxidase Activity

Peroxidase activity was measured according to the method reported by Morales-Blancas et al. (2002). Peroxidase substrate solution was prepared daily by mixing 0.1 mL guaiacol, 0.1 mL hydrogen peroxide (30%), and 99.8 mL potassium phosphate buffer (0.1mol/L, pH 6.5). Peroxidase assays were conducted by pipetting 0.12 mL of enzyme extract and 3.48 mL of substrate solution in the 10 mm path-length quartz cuvette. Peroxidase activities were measured from the increase in absorbance at 470 nm using an UV/vis spectrophotometer (UV-mini 1240, Shimadzu, Japan). The reaction was monitored for 20 min at 5sec intervals at 25ËšC. Enzyme activity was calculated from the slope of the initial linear portion of a plot of absorbance vs. time. All the experiments were replicated thrice. Residual enzyme activity (REA) in heat-treated samples is expressed as a fraction of initial activity (C0):

Residual enzyme activity (REA) = C/C0 Ã-100

Where C and C0 are ∆Abs./min after heat treatment for time t and native enzyme, respectively.

Measurement of Colour

Colour analyses were carried out on fresh and heat treated seedless guava using a tristimulus colorimeter (Minolta Chroma Meter, Osaka, Japan) equipped with a CR-300 measuring head in terms of L-value (lightness), a-value (redness and greenness), and b-value (yellowness and blueness) as an average of three measurements at three different locations. The instrument was standardized against a white tile before measurements (L = 97.67; a = 0.08 and b = 1.54).

From these values, total colour difference (TCD) was calculated according to the following equations:

Where L0, a0 and b0 are the readings at time zero, and L, a and b the individual readings at each processing time.

To minimize the variability between different raw samples, the individual L, a and b values were normalised, dividing the parameters by the corresponding initial values (Cruz et al. 2007).

Calculation of Kinetic Parameters

The zero- (Eq. (3)) and first-order (Eq. (4)) equations were used to describe the enzyme inactivation and colour changes in seedless guava:

Where C is the value of peroxidase activity and colour parameters at time t, C0 is the initial value at time zero, k is the rate constant at the process temperature, and t is time. The plot of the logarithm of the ratio C/C0 against time would yield a straight line, with the rate constant equal to the negative of the slope.

The temperature dependence of the rate constant is normally described by an Arrhenius Law:

Where Ea is the activation energy, R is the gas constant (8.314 Jmol-1 K-1) and T is the temperature in °K.

The time-dependent kinetic parameter can also be expressed as the D-value (or decimal reduction time), which is the time required for a decimal change in the property value at constant temperature. The D-value is inversely related to the first-order reaction constant (k):

Statistical Analysis

Data processing was performed using StatSoft, Inc. (2001) STATISTICA-data analysis software system-version 6. A zero and first order kinetics model was considered to follow Eq. (3) and Eq. (4). The zero and first order kinetics fit was also obtained using the simple regression option of the General Linear Model. The parameters Ea and D were similarly obtained from the Arrhenius and reduction decimal Eqs. (5) and (6), respectively.

An analysis of variance (one-way ANOVA with replication) using the Tukey test was performed to assess the thermal process time-temperature conditions effects on peroxidase activity and colour changes. Differences between means were considered to be significantly different at P < 0.05. Parameters' precision was evaluated by confidence intervals at 95%, and the quality of the regression was assessed by the coefficient of determination (R2).


Kinetics of Peroxidase Inactivation

Peroxidase is generally one of the most thermally stable enzymes found in fruits and vegetables. Although the role of peroxidase in causing quality changes is not well established, it is a commonly used indicator for the inactivation of endogenous enzymes during heating because the assay is simple and rapid. Inactivation kinetics for peroxidase from seedless guava has not been previously reported. The seedless guava cubes were thermally treated by using the hot water in the temperature range of 80-95° C.

Fig. 1 shows the residual peroxidase activities (in %) as a function of time at four different temperatures. The results clearly show that the amount of inactivation increases with increasing temperature and treatment time. Inactivation time and temperature had significant (P < 0.05) effect on the inactivation of seedless guava peroxidase and there was a significant interaction between inactivation time and temperature (data not shown).


The semi-log plot of the residual activity of peroxidase (Fig. 2) versus heating time were linear at all temperatures studied, which is consistent with inactivation by means of a simple (monophasic) first-order process. Monophasic behavior of the enzyme inactivation at high temperatures could be due to the rapid inactivation of the heat-labile fraction of the enzyme during the first seconds of treatment, so the observed kinetics would correspond to the inactivation of the heat-resistant fraction of peroxidase.

This result is in accordance with those obtained for peroxidase from pepper (Serrano-Martínez et al. 2008), potato or carrot (Anthon and Barrett, 2002) carrots, potatoes, tomato, green beans, green asparagus and pumpkin (Soysal and Soylemez 2005; Anthon and Barrett 2002; Anthon et al. 2002; Ganthavorn et al. 1991; Goncalves et al. 2007), but contrasts with those obtained for potato and carrot polyphenolxidase (Anthon et al. 2002). From the slopes of these lines, the inactivation rate constants (k) were calculated. The k values increased with temperature from 1.2(±0.0001)Ã-10-2 at 80°C to 4.5(±0.0004)Ã-10-2 s-1 at 95°C.

The Arrhenius plot for peroxidase inactivation over a temperature range of 80-95 °C is shown in Fig. 3. It illustrates the temperature dependence of peroxidase inactivation (80-95 °C). Activation energy of peroxidase inactivation were found from the slopes of the curves as 96.39±4 kJmol-1 (R2 = 0.9989). Decimal reduction times of peroxidase were also determined (Table 1).


[Fig 3]

Three main processes have been considered to be involved in the inactivation of peroxidase; (1) dissociation of prosthetic (heme) group from the haloenzyme (active enzyme system); (2) conformation change in the apoenzyme (protein part of the enzyme); and/or (3) modification or degradation of the prosthetic group (Lemos et al. 2000).

According to obtained activation energy (Table 1), the observed rate reflects the loss of some functional group or the dissociation of the heme group in the case of peroxidase that need lower activation energy among other proposed processes.

[Table 1]

Kinetics of Colour Changes

Figs. 4, 5, 6 and 7 illustrate the changes of L, a, b and total colour difference (TCD) of thermally treated seedless guava. During thermal treatments, L, a and b values decreased with an increase in temperature and processing time (P < 0.05), suggesting that seedless guavas loss greenness, yellowness and become darker.

Enzymatic browning is a serious problem because the oxidative enzymes, such as peroxidase and polyphenolase, may cause browning accompanied by changes in colour, flavor and nutritive value (Reyes and Luh 1960). During thermal treatment, those enzymes were inactivated, but many other reactions can take place affecting colour. Chlorophyll and carotenoid pigments decomposition (Kostaropoulos and Saravacos 1995; Lee and Coates 1999; Weemaes et al. 1999) and formation of brown pigments by non-enzymatic browning (Millard) reactions are the most common (Rhim et al. 1989; Lopez et al. 1997).

In the present study, variation of colour parameters L, a, b with the treatment time at different temperatures were well fitted to a first order kinetic (R2= min- 0.9816, max-0.9965). Barreiro et al. (1997), Avila et al. (1999) and Ahmed et al. 2002 applied the same kinetic model for apple, peach, prune purees and tomato paste treated thermally.

In contrast, changes of total colour difference (TCD) was found to follow a zero order kinetic reaction model (R2= 0.9872). TCD is one of the best parameters for describing the colour variation since it is a combination of parameters L, a and b. Aguerre and Suarez (1987); Rhim et al. (1989) and Barreiro et al. (1997) applied the same kinetic model for corn, grape juice, double concentrated tomato paste treated thermally. The rate constants (k) and decimal reduction times (D) of color parameters changes are reported in Table 2.

The Arrhenius plot for colour parameters changes over a temperature range of 80-95 °C is shown in Fig.8 A-D. The activation energies of L, a, b and TCD were estimated as the slopes of lines and are shown in Table 2. Activation energies are 122.68±3 kJmol-1, 88.47±5 kJmol-1, 104.86±5 kJmol-1 and 112.65±5 kJmol-1 for L, a, b and TCD values, respectively. The lower activation energies for a value indicated that a was less sensitive than L and b at all temperatures.

[Fig 4]

[Fig 5]

[Fig 6]

[Fig 7]

[Fig 8]

[Table 2]


The effect of thermal treatments on the activity of the enzyme peroxidase in seedless guava (Psidium guajava L.) was studied. At the range of temperatures (80 - 95°C), the heat-labile fraction was rapidly inactivated and the kinetic behavior corresponds to the heat-resistant fraction that followed monophasic first-order inactivation kinetics. The colour changes were well described by zero- and first-order kinetic model. The Arrhenius model described the temperature dependence of the reaction rate constant of all the factors considered. With these models and the estimated kinetic parameters, it would be possible to predict the residual peroxidase activity and colour changes for a given set of time-temperature conditions. This research study will help to design the seedless guava (Psidium guajava L.) blanching conditions.