1794
J. Agric. Food Chem. 1999, 47, 1794−1800
Kinetic Study of the Irreversible Thermal and Pressure Inactivation of Myrosinase from Broccoli (Brassica oleracea L. Cv. Italica) L. Ludikhuyze, V. Ooms, C. Weemaes, and M. Hendrickx* Department of Food and Microbial Technology, Faculty of Agricultural and Applied Biological Sciences, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Heverlee, Belgium
Thermal and pressure inactivation of myrosinase from broccoli was kinetically investigated. Thermal inactivation proceeded in the temperature range 30-60 °C. These results indicate that myrosinase is rather thermolabile, as compared to other food quality related enzymes such as polyphenol oxidase, lipoxygenase, pectinmethylesterase, and peroxidase. In addition, a consecutive step model was shown to be efficient in modeling the inactivation curves. Two possible inactivation mechanisms corresponding to the consecutive step model were postulated. Pressure inactivation at 20 °C occurred at pressures between 200 and 450 MPa. In addition to its thermal sensitivity, the enzyme likewise is rather pressure sensitive as compared to the above-mentioned food quality related enzymes. By analogy with thermal inactivation, a consecutive step model could adequately describe pressure inactivation curves. At 35 °C, pressure inactivation was studied in the range between 0.1 and 450 MPa. Application of low pressure (90% after a treatment at 70 °C for 30 min (Yen and Wei, 1993). The higher temperatures reported for myrosinase inactivation in these cases may be due to limitations in heat transfer involved in the heat treatment of whole vegetables as compared to vegetable enzyme extracts. First, the validity of the first-order kinetic model (Table 1), which is frequently reported in the literature to describe enzyme inactivation (Suzuki, 1960; Lopez and Burgos, 1995; Ludikhuyze et al., 1997, 1998a), was evaluated. Because regression coefficients varied between 0.413 and 0.845 and residuals were not randomly distributed, it was concluded that a first-order kinetic model could not describe the irreversible thermal inactivation of myrosinase. Hence, the appropriateness of three other mathematical models (Table 1), likewise suggested in the literature for modeling enzyme inactivation (Robert et al., 1995; Weemaes, 1998), has been evaluated. A two-isozyme model refers to the existence of two isozymes, behaving differently when subjected to heat. This results in an initial fast inactivation period, followed by a much slower decay, eventually leading to a plateau phase. A consecutive step model can be used
Table 3. Fractional Conversion Model: Estimation of the Kinetic Parameters for Thermal Inactivation of Myrosinase from Broccoli (0.015 g of Lyophilized Powder/mL in 0.1 mol/L Phosphate Buffer at pH 6.55) k (min-1)
T (°C)
Afa
30 35 37.5 40 42.5 45 50 60
89.5 ( 80.7 ( 1.3 53.4 ( 3.1 51.7 ( 1.7 41.3 ( 2.5 35.8 ( 3.5 23.5 ( 2.7 4.5 ( 0.9
(3.9 ( 1.8b) × 10-2 (1.2 ( 0.2) × 10-1 (1.3 ( 0.3) × 10-1 (2.1 ( 0.3) × 10-1 (2.6 ( 0.4) × 10-1 (4.2 ( 1.0) × 10-1 (5.5 ( 1.1) × 10-1 (24.0 ( 3.0) × 10-1
1.5b
a Given in percent of the initial overall activity. b Standard error.
to describe two different cases of enzyme inactivation. (i) The consecutive step model can describe an inactivation process proceeding as a succession of two irreversible reaction steps: k1
k2
N 98 I 98 D In the first step, the native enzyme converts to an intermediate form, the activity of which may be dependent on the inactivation temperature applied. On its turn, this intermediate form is converted to the inactive form. (ii) The consecutive step model can likewise be used to describe the inactivation of enzymes that exist as two different forms (e.g., free enzyme and enzyme interacting with impurities or substrates) which are in equilibrium with each other.
The transformation of each enzyme form to the inactive form is characterized by a distinct inactivation rate constant. Clearly the position of the equilibrium between the two forms can be dependent on temperature. The fractional conversion model, on its turn, refers to a first-order inactivation process in which a resistant enzyme fraction, not affected after prolonged heating at the preset temperature, is taken into account. Calculation of the kinetic parameters corresponding to the mathematical equations proposed was accomplished by fitting the data to eqs 8, 9, and 10, respectively, using a nonlinear regression procedure (SAS Institute, 1982). Concerning the two-isozyme model, activity estimates of both the native thermostable (As) and thermolabile (Al) enzyme forms are presented in Table 2, showing both As and Al to vary with temperature. Activity of the native thermostable form decreased with increasing
Inactivation Kinetics of Myrosinase from Broccoli
J. Agric. Food Chem., Vol. 47, No. 5, 1999 1797
Table 4. Consecutive Step Model: Estimation of the Kinetic Parameters for Thermal Inactivation of Myrosinase from Broccoli (0.015 g of Lyophilized Powder/mL in 0.1 mol/L Phosphate Buffer at pH 6.55) 30 35 37.5 40 42.5 45 50 60 a
k1 (min-1)
A2a
k2 (min-1)
NC (1.4 ( 0.4c) × 10-1 (3.3 ( 0.9) × 10-1 (2.4 ( 0.7) × 10-1 (4.2 ( 0.7) × 10-1 (9.8 ( 0.2) × 10-1 (9.3 ( 0.1) × 10-1 (27.9 ( 1.7) × 10-1
NC 81.6 ( 3.3b 70.2 ( 2.7 55.7 ( 4.6 52.5 ( 2.6 49.5 ( 1.3 34.1 ( 2.1 7.9 ( 0.7
NC (4.0 ( 0.4c) × 10-4 (1.8 ( 0.2) × 10-3 (4.9 ( 0.7) × 10-3 (6.1 ( 1.2) × 10-3 (1.2 ( 0.2) × 10-2 (1.5 ( 0.2) × 10-2 (3.5 ( 0.7) × 10-2
A1a
T (°C)
NCb
101.2 ( 1.6c 99.7 ( 2.7 99.9 ( 3.6 98.6 ( 2.4 99.6 ( 3.8 99.9 ( 2.5 99.9 ( 0.7
Given in percent of overall initial activity. b NC, no convergence. c Standard error.
Table 5. Statistical Analysis of the Fractional Conversion Model (FCM) and the Consecutive Step Model (CSM) To Select the Model Most Adequately Describing Thermal Inactivation of Myrosinase from Broccoli T (°C) 30 35 37.5 40 42.5 45 50 60
model
corrected r2
s2 (×104)
AOC
SOC
FCM CSM FCM CSM FCM CSM FCM CSM FCM CSM FCM CSM FCM CSM FCM CSM
0.9997
2.77
-38.7
-55.8
0.9997 0.9997 0.9927 0.9986 0.9976 0.9971 0.9945 0.998 0.9847 0.9957 0.9558 0.9973 0.996 0.9996
2.37 3.16 39.2 7.51 12.4 14.5 23.2 6.49 51.5 14.2 34.2 6.24 4.95 0.55
-49.1 -22.0 -37.7 -42.0 -42.7 -25.1 -37.0 -32.3 -29.9 -25.3 -27.7 -16.6 -43.2 -35.9
-64.9 -61.7 -53.1 -67.8 -58.1 -55.3 -52.4 -62.6 -45.3 -55.5 -43.6 -56.3 -59.0 -75.7
temperature while that of the native thermolabile enzyme increased. This inconsistency of the initial activity estimates indicated a lack of evidence for the presence of two fractions differing in thermostability. Indeed, in this case the activity estimates should be independent of temperature because they are the initial activities of two native isozymes. Consequently, the twoisozyme model was rejected. Subsequently, the remaining models, that is, the fractional conversion model (Table 3) and the consecutive step model (Table 4), were evaluated and statistically compared by calculating s2, AOC, and SOC (Table 5). Table 3 indicates the activity estimates of the hypothetical intermediate (A2) to vary significantly with temperature. However, exclusion of the consecutive step model based on this observation is not justified because the activity of the intermediate or the position of the equilibrium between two enzyme forms may depend on the inactivation temperature (Robert et al., 1995). From a statistical point of view, the fractional conversion model is preferred at temperatures
