Kinetic Characterization and Thermal Inactivation of Peroxidase in Aqueous Extracts from Sweet Corn and Waxy Corn
The objective of this study was to evaluate the activity, kinetic behavior, and thermal inactivation kinetics of peroxidase (POD) in aqueous extracts from two kinds of milk ripe stage corn, sweet corn and waxy corn. Optimum activities using guaiacol as the hydrogen donor were obtained for sweet corn at pH 4.8 and for waxy corn at pH 6.0. The kinetics of POD showed characteristics which were dependent upon the concentrations of guaiacol and H2O2. The guaiacol Km values for sweet corn POD and waxy corn POD were 11.01 and 23.01 mM, respectively, whereas the H2O2Km values for sweet corn POD and waxy corn POD were 2.85 and 0.33 mM, respectively. Thermal treatment of enzymatic aqueous extracts was carried out at different time–temperature combinations in the range of 0–25 min and 60–85 °C. Arrhenius plot determination and calculated thermodynamic parameters suggested that the inactivation of POD followed first-order reaction kinetics, and the activation energy (Ea) for inactivation of sweet corn POD (114.36 kJ/mol) was slightly lower compared with waxy corn POD (119.72 kJ/mol). There were several notable similarities between the inactivation kinetics in the two corn cultivars.
KeywordsPeroxidase Sweet corn Waxy corn Kinetic characterization Thermal inactivation
Corn (Zea mays L.), also called maize, is readily available as fresh, canned, or frozen and largely consumed worldwide due to its soft grains, thin shells, and tastefulness (Scott and Eldridge 2005). Sweet corn (Z. mays L. ssp. saccharata Sturt) is a variety of maize with a high sugar content, which is usually picked when immature (milk stage), prepared and eaten as a vegetable, rather than a grain. Waxy corn (Z. mays L. var. ceratina Kulesh), whose endosperm contained only amylopectin and no amylose starch molecule, tastes stickier than other maize varieties. When raw or processed corn is held in frozen storage for extended periods of time, quality deterioration may occur, such as loss of nutrients, undesirable color changes, off-flavor development, and softening of the texture. A high correlation between the quality changes and enzymatic activity was confirmed (Lee and Hammes 1979; Garrote et al. 1987).
Blanching vegetables to inactive endogenous enzymes is a critical step prior to freezing (Barrett et al. 2000; Gonçalves et al. 2007). Among various enzymes, peroxidase (POD) has been the most popular indicator enzyme in the blanching process because of its high concentration in most plant tissues, high thermal stability, and ease of assay (Burnette 1977; Anthon and Barrett 2002; Gonçalves et al. 2010). POD is an oxidoreductase that is widely distributed in nature, whose primary function in plants is the reduction of H2O2 at the expense of oxidation of phenolic compounds (Połata et al. 2009). Normally, POD does not exist as a single enzyme, but as isoforms in plants; hence, its activity is expressed in the form of a number of discrete isoenzymes. Also, POD exists in plant cells in both soluble and membrane-bound forms (Anthon and Barrett 2002), with the former accounting for a higher proportion in most vegetables, suggesting that studies on the thermal stability of POD in blanched and frozen vegetables should focus mainly on the soluble fraction (Morales-Blancas et al. 2002). In particular, some processed products do not require complete inactivation of POD in order to maintain color, texture, flavor, and nutritional quality. Moreover, reduced blanching time would benefit the industry by decreasing energy costs, water use, and clean up costs. The control of blanching to keep POD inactivation to a suitable residual level is important, emphasizing that the need for the analysis of the kinetics of the process in plants has been recommended (Bahçeci et al. 2005).
Because of the high thermostability, the involvement of POD in thermal inactivation in sweet corn has been studied extensively. Yamamoto et al. (1962) investigated heat inactivation over a wide range of temperatures and showed that inactivation of POD in whole kernel corn was biphasic, suggesting the presence of heat-sensitive and heat-resistant fractions. The latter represented 5 % of total enzyme activity and was concentrated in the pericarp. Lee and Hammes (1979) found that POD residual activity in the outer cob and kernels of corn-on-the-cob blanched in steam for variable times had significant correlations with off-flavor development in corn-on-the-cob. Luna et al. (1986) developed a thermokinetic model describing POD inactivation during blanching–cooling of corn-on-the-cob, which allowed the calculation of the POD activity retention for the kernel, outer cob, and central cob. Collins et al. (1996) suggested that the different POD bandings among different sweet genotypes might be involved in their different performance of flavor quality during frozen storage, and Barrett et al. (2000) evaluated POD activity of three corn cultivars which behaved differently as a function of blanching time.
For successful predictions of the residual enzyme activities to minimize the loss of nutritional and sensory properties, it is necessary to know the enzyme distribution and the parameters of the inactivation kinetics; this information is unique for each vegetable, specie, cultivar, and environmental conditions (Agüero et al. 2008). No published information is available in literature on thermal inactivation of POD during blanching of waxy corn, not to mention the differences between sweet corn and waxy corn. Therefore, the objective of the present study was to study the activity and kinetics of POD from two corn cultivars and to examine the thermal inactivation kinetics. The results will help to optimize blanching process for corn, which will certainly be important for corn products development. Non-purified enzyme extract was chosen in order to obtain information relevant to fresh corn processing at the industrial level. The results were also compared with published data for previously studied plant POD.
Materials and Methods
Sweet corn cobs and waxy corn cobs, at the milk ripe stage as determined by moisture and consistency of the kernel parenchyma, were obtained in a local market in Nanjing, China. Corn samples were stored at 4 °C in husk and all experiments were carried out within 48 h.
Hydrogen peroxide (30 %) and guaiacol (99.5 %) were purchased from Sinopharm Chemical Reagent Co. Ltd., China. Sodium phosphate buffer (pH 7.0) was prepared with sodium dihydrogen phosphate and disodium hydrogen phosphate in distilled water obtaining a molar concentration of 0.2 mol/L, the buffer solution was cooled at 4 °C until used. All other chemicals and reagents were of analytical grade. Distilled water was used for all assays.
Extraction and Determination of Crude POD Activity
The intact kernels and germ fraction were separated manually, about 5.0 g samples was homogenized in ice-water bath with 25 mL phosphate buffer (pH 7.0, 4 °C), then centrifuged at 4 °C, 10,000×g for 30 min, the supernatant was collected and divided into 2 mL each sample, and then the samples were quickly cooled in liquid nitrogen for analysis of POD enzyme activity. The POD activity was determined by measuring the increase of absorbance at 470 nm (ε470 = 26.6 mM−1 cm−1) using guaiacol as a phenolic substrate with hydrogen peroxide (Díaz et al. 2001). The reaction mixture contained 0.5 mL of 1.5 % (m/v) guaiacol, 0.2 mL of 1 % (v/v) H2O2, 2.1 mL of 0.1 mol/L phosphate buffer (pH 6.0), and 0.2 mL of the enzyme extract. The blank sample contained the same mixture solution without the enzyme extract. The maximal initial reaction velocity was calculated over 20 to 140 s linear increase in absorbance. One unit of enzymatic activity was defined as amount of enzyme that oxidizes 1 mM of guaiacol per minute at 25 °C and pH 6.0 under assay conditions.
Effect of pH on Enzyme (Extracted from Corn Kernels) Activity
To determine the pH optimum for POD, activity measurements were carried out over a pH range of 3.0 to 9.0. The three buffers used were citrate phosphate (pH 3.0–5.5), sodium phosphate (pH 6.0–7.5), and borate (pH 8.0–9.0). POD activity was measured according to the method described above and expressed as a percentage of the maximum activity.
In order to determine the kinetic properties, POD (extracted from corn kernels) activity was evaluated toward guaiacol concentrations ranging from 1.0 to 20.0 mM and H2O2 concentrations ranging from 0.5 to 10.0 mM. The Michaelis–Menten constants (Km) were determined from Lineweaver–Burk plots at optimum pH conditions.
Thermal Inactivation Experiments
The thermal inactivation of crude POD extracts from sweet corn kernels and waxy corn kernels was studied at constant temperature between 60 and 85 °C at atmospheric pressure. Aliquots of the homogenates were transferred to centrifuge tubes, each tube with 1.0 mL of enzyme extracts. Samples were heated in a circulating water bath to the indicated temperatures for the times specified. The temperature of the water bath was controlled to 0.1 °C. Following heating, the samples were cooled in ice-water to stop thermal inactivation instantaneously and stored on ice until assay.
All determinations were conducted three times at least. Regression analysis was made by using Microcal Origin 8.5 (Microcal Software, Inc., Northampton, USA). One-way analysis of variance was determined using the Tukey–Krammer test. Differences between means were considered to be significantly different at P < 0.05.
Results and Discussion
POD Distribution in Fresh Corns
POD activity in sweet corn and waxy corn
POD activity (U)
11.89 ± 0.14a
3.36 ± 0.23
5.59 ± 0.37
7.12 ± 0.56
Effect of pH on POD Activity
In addition, a similar approach was then taken to determine the kinetic parameters for H2O2 for both corns at 20 mM guaiacol. As shown in Fig. 2c, d, when the H2O2 concentration was increased, the activity increased to reach saturation at 3.5 mM for sweet corn and 5.0 mM for waxy corn, and then decreased with H2O2 concentration above the saturation. Hence, these did not follow pure Michaelis–Menten kinetics. The forms of these graphs were typical of enzymes exhibiting substrate inhibition. At the same time, it was found that the Km values for H2O2 calculated by Lineweaver–Burk reciprocal plots was identical to that estimated from the substrate saturation curves, Km values were 2.85 and 0.33 mM for sweet corn and waxy corn, respectively (Fig. 2c, d, inset). Noticeably, the Km value of waxy corn was eight times lower than sweet corn, which implied that waxy corn POD had a relatively high affinity for H2O2. Other reported Km values for H2O2 by using guaiacol include 7.2 mM for green pea (Halpin et al. 1989), 1.4 mM for carrot (Soysal and Söylemez 2005), and 50.68 and 18.18 mM for two different strawberry cultivars (Chisari et al. 2007), but no inhibition by substrate concentration was observed between them.
Thermal Inactivation Kinetics of POD Crude Extracts
Thermal inactivation parameters for POD in sweet corn and waxy corn
0.18 ± 0.02a
12.76 ± 1.43
0.37 ± 0.01
6.22 ± 0.20
0.77 ± 0.03
2.98 ± 0.12
1.27 ± 0.20
1.82 ± 0.27
3.26 ± 0.25
0.71 ± 0.06
114.36 ± 6.97
20.08 ± 0.09
0.10 ± 0.01
24.27 ± 1.58
0.26 ± 0.03
8.91 ± 0.93
0.54 ± 0.07
4.27 ± 0.53
0.88 ± 0.13
2.61 ± 0.51
1.18 ± 0.13
1.96 ± 0.26
119.72 ± 5.11
18.38 ± 0.44
The present results indicated that POD activity in aqueous extracts from sweet corn and waxy corn differed significantly in total kernel or germ fraction and varied by pH and substrate concentrations. The enzyme kinetics followed Michaelis–Menten equation, showing different values of POD kinetics parameters between the two cultivars. In addition, the thermostability of POD is very important due to its negative effect on the color and flavor of corn during storage. POD from sweet corn and waxy corn showed diverse heat sensitivities with variations in K, Ea, D, and ZT. Taking those values into consideration, POD from waxy corn was found to be more heat stable than sweet corn, which implies that the blanching parameters should be optimized discriminately.
The authors would like to thank the financial support from Jiangsu Academy of Agricultural Sciences for Innovation in Agriculture Science and Technology [project no. CX (11) 4026].
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