measurement of the production rate of methanol; one of the products of the PME ... The kinematic viscosity of the food model solutions was measured using ...
Med. Fac. Landbouww. Univ. Gent, 66/4, 2001
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KINETICS OF ENZYME CATALYSED REACTIONS IN FROZEN FOOD MODEL SYSTEMS AND GLASS TRANSITION THEORY NETSANET SHIFERAW TEREFE, MINH TRI NHAN, MULUGETA ADMASU DELELE, JOSHUA ARMI, ANN VAN LOEY, MARC HENDRICKX Laboratory of Food Technology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 22, B3001, Leuven
INTRODUCTION Glass transition theory relates the stability of frozen foods with the physical state of the unfrozen matrix surrounding the ice crystals within the frozen food (Levine and Slade, 1989). According to this theory, the translational mobility of molecules is reduced to practical insignificance below the glass transition temperature of the maximally freeze-concentrated phase (Tg’) so that the rate of diffusion limited chemical and physical processes will be greatly reduced. We studied the kinetics of two enzyme catalyzed reactions namely the alkaline phosphatase catalyzed hydrolysis of disodium-p-nitrophenyle phosphate and the pectin methylesterase (PME) catalyzed de-esterification of pectin in frozen model systems. In both cases no consistent relationship between the kinetics and Tg’ was observed (Terefe et al, 2002; Terefe and Hendrickx, 2002). The purpose of this study was to examine the reasons for the observed inconsistency. Thus the two reactions were further investigated in the above model systems varying the macroviscosity of the medium by varying solute concentration and temperature, in order to ascertain whether they are diffusion controlled. The result of the PME catalyzed reaction is presented. MATERIALS AND METHODS
Experimental protocol for the kinetic study The kinetic study on the PME catalysed de-esterification of pectin was performed as described in detail in Terefe and Hendrickx (2002). The assay used is based on the measurement of the production rate of methanol; one of the products of the PME catalysed de-esterification of pectin. The concentration of methanol was measured using the method of Klavons and Bennet (1986). Tomato PME (EC 3.1.1.11, Sigma, Bornem, Belgium) with an activity of 67units/mg and apple pectin (Fluka, Bornem, Belgium) with 70-75% degree of esterification were used in this study. A pectin solution (7g/l) was prepared in a buffer (0.1M of Tris (Hydroxymethyl)-methane, Merck, Damstadt, Germany). The pH of the substrate solution was adjusted to 7.5 by adding 7.5ml/l of 37% HCl solution. An enzyme solution (0.12mg/ml) was prepared in distilled water. 20%(w/w) sucrose (Sigma), 20% (w/w) fructose (Sigma), 20%(w/w) maltodextrin with DE=16.5-19.5 (Aldrich, Milwaukee, WI, USA) and
2 5% (w/w) low viscosity sodium carboxymethylcellulose (CMC) (Sigma) were prepared in distilled water. Each of these solutions was mixed with an equal portion of the substrate solution to be used as food model in the kinetic study. The initial rate Vo was taken as the initial slope of the methanol concentration versus time curve. Regression analyses on the data were performed using SAS statistical software (SAS release 6.12, SAS Institute Inc., Cary, NC, USA). Determination of Tg’ Differential scanning calorimetery (DSC) was used to measure the glass transition temperature of the food model solutions. Perkin-Elmer DSC-7 equipped with a liquid nitrogen-cooling accessory was used in the measurement. ± 20mg of sample solution encapsulated in aluminium pan was scanned against an empty reference pan at rate of 10°C/min both during cooling and reheating and the Tg’ was determined from the heating scan. Experiments to determine if the reaction is diffusion controlled The classical method to determine the extent to which a reaction is diffusion controlled is to investigate the dependence of the observed rate of reaction upon increasing concentration of a viscogenic agent. For a bimolecular reaction: k1 A+B
k2 A.B
P
(1)
k-1 Assuming spherical molecules and the Stokes-Einstein equation applicable r r 2RT k1 2 A B (2) 3η rB rA : viscosity of the medium, rA, rB: hydrodynamic radius of the diffusing molecule. The global rate constant k1k 2 K (3) k 1 k2 When diffusion is rate limiting (k2 >> k-1), K = k1 (4) From (2) and (4) Ko η (5) K ηo o is a reference; a substrate solution without added viscogen. The kinetics of the PME catalysed de-esterification of pectin was studied at 25°C in the model systems by varying the concentrations of the added solutes from 0 to 70%(m/v). Kinetic experiment was also performed in two concentrations of maltodextrin and sucrose model systems in a temperature range of –4 to 20°C.
Med. Fac. Landbouww. Univ. Gent, 66/4, 2001
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Viscosity measurement The kinematic viscosity of the food model solutions was measured using Ubbelhode capillary viscometers (Schott glaswerke, Mainz, Germany). The density was measured with pycnometer. The dynamic viscosity was obtained from the product of the kinematic viscosity and the density.
RESULTS AND DISCUSSION The glass transition temperatures of the different model systems are given in table 1. As can be seen, the presence of the substrate buffer has a significant effect on the value of Tg’. Table 1. The glass transition temperatures (°C) of the model systems Material
pure
CMC fructose Maltodextrin (DE= 16.5-19.5) sucrose
-13.07 -42 -12.5 -31.5
With substrate buffer -19.1 -44.1 -21.3 -35.1
0.4
130
0.35
110
0.3
90
0.25
70
0.2 50
0.15
30
0.1
10
0.05 0
0
20
40
60
[mpa.s]
90 80 70 60 50 40 30 20 10 0
K[l/U.hr]
Vo[µg methanol/ml.hr]
The initial rate of de-esterification of pectin in the different models systems versus T-Tg’ is presented in figure 1. In all the model systems, the reaction rate decreased to a very low value far above the respective glass transition temperature.
-10
T-Tg'[°C]
0 20 40 60 80 solute concentration[% m /v]
Fig. 1. The kinetics of the PME catalysed de-esterification of pectin in four frozen model systems (: CMC, :fructose, : maltodextrin, : sucrose).
Fig. 2. The global rate constant (K) for the PME catalyzed de-esterification of pectin and at 25°C versus concentration for three model systems (: fructose, : maltodextrin, : sucrose)
The global reaction rate constant, K, for the PME catalysed de-esterification of pectin versus solute concentration for all the model systems except CMC is shown in figure 2. The viscosity data is also presented on the same figure. In the case of
4 CMC, an increase in reaction rate followed by a decrease with solute concentration was observed probably due to the presence of the sodium ion in the CMC system, which is an activator of PME (data not shown). The monotonic decrease in the reaction rate with solute concentration in the other systems indicates that the reaction is diffusion controlled. However the expected inverse relationship between the relative reaction rate constant and the relative viscosity (according to equation 5) was not observed as is shown for the sucrose model system in figure 3. This suggests that the diffusion of the reactants may not be controlled by the macroviscosity of the system. 7
0 0.0033 0.0034 0.0035 0.0036 0.0037 0.0038 -0.5
5
-1
4
-1.5 lnK
Ko/K
6
3
-2 -2.5
2
-3 1
-3.5
0 0
10
20
30
40
Fig 3. Relative reaction constant versus relative viscosity for the PME catalyzed de-esterification of pectin in sucrose model system
-4 1/T[K]
Fig. 4 Arrhenius plots for the kinetics of the PME catalyzed de-esterification of pectin in four model systems (: 47% sucrose, : 30% sucrose, : 47% maltodextrin, : pectin)
Arrhenius plots for the kinetics in different model systems and the pure substrate solution are shown in figure 4. The same slope (activation energy) was observed in all cases indicating that the underlying fluid phase viscosity is the same. The difference in the pre-exponential factor is most probably due to the difference in the degree of obstruction to the molecular mobility of molecules presented by the solutes in the dispersed phase. Thus the concentration and type of the solutes comprising the model matrix, which affect the extent of the obstruction effect, and the solvent viscosity seem to be the main factors that control the kinetics of the reaction investigated rather than Tg’. REFERENCES Levine, H., Slade, L. (1989). A food polymer science approach to the practice of cryostabilization technology. Comments agric. Food Chem. 1(6), 315-396. Terefe, N.S., Hendrickx, M. (2002). Kinetics of the pectin methylesterase catalysed de-esterification of pectin in frozen model systems. Biotechnol. Prog. 18(2), 221-228. Terefe, N.S., Mokwena, K.K.; Van Loey, A., Hendrickx, M. (2002) Kinetics of the alkaline phosphatase catalysed hydrolysis of disodium p-nitphenyl phosphate in frozen model systems. Biotechnol. Prog. 18(6), 1249-1256. Klavons, J.A., Bennet, R. D.(1986). Determination of methanol using alcohol qxidase and its application to methyl ester content of pectins. J. Agric. Food Chem. 34, 597-599.
ACKNOWLEDGEMENT This research has been supported by the Interfaculty Council for Development Co-operation of the Katholieke Universiteit Leuven.
