Journal of Food Engineering 39 (1999) 167177

Activity of pectin methyl esterase during blanching of peaches

L. M. M. Tijskens a,*, P. S. Rodis b, M. L. A. T. M. Hertog a, N. Proxenia b, C. van Dijk a
a

ATO-DLO, P.O. Box 17, Bornsesteeg 59, 6700 AA Wageningen, The Netherlands b Agricultural University of Athens, Greece Received 10 April 1998; accepted 18 November 1998

Abstract The activity of pectin methyl esterase (PE) in peaches during blanching treatments was modelled and analysed. It was postulated that the enzyme exists in two congurations, one bound and one soluble. The bound conguration can be converted into the soluble conguration. These two congurations have a dierent susceptibility to temperature. All rate constants of reaction were modelled as dependent on temperature according to the Arrhenius law. Despite the daily measuring variance, the variance accounted for by the model in multivariate nonlinear regression analysis was more than 90%. The obtained parameter values were highly comparable for two consecutive seasons. An analysis of the data of the two seasons combined was feasible with the kinetic parameters estimated in common, without losing information with a tremendous increase in predictive power. 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Enzyme denaturation; Enzyme conversion; Kinetics; Apparent activity; Blanching; Peaches; Generic model

Nomenclature E k PE R t T Nobs R2 adj Indices abs c d x i na ref tot var 0 1 2

activation energy, J/mol reaction rate constant, s1 activity of PE, nkatal/gFWT universal gas constant, J/K/mol time, s temperature, C or K number of observations explained part, %

absolute temperature conversion denaturation xed part any not active at reference temperature total variable part initial bound conguration soluble conguration

1. Introduction The aim of blanching fruits and vegetables prior to sterilisation is among others, the activation and/or in* Corresponding author. Tel.: +31-317-475-303, fax: +31-317-475347; e-mail: l.m.m.tijskens@ato.dlo.nl

activation of enzymes present in the plant tissue (Pilknik & Voragen, 1991). The apparent activity of dierent enzymes at dierent temperatures exhibits a well-known behaviour: at temperature below 50C, a steady increase in activity with increasing temperatures is observed. This increase in apparent activity can frequently be described by the Arrhenius law. Eventually, a maximum activity is reached. This is often referred to as the optimal temperature for enzymatic action. At still higher temperatures, a rather steep decline in activity is observed (Segel, 1993; Whitaker, 1994; Wiley, 1994) due to denaturation. The action of these enzymes, such as pectin methyl esterase, polygalacturonase and peroxidase, will have a pronounced eect on the observed quality of fresh and processed products. The enzyme pectin methyl esterase (PE) can be found in many fresh fruits and vegetables (Rexova-Benkova & Markovic, 1976). The action of the enzyme comprises the demethylation of the carboxymethyl groups of pectic polysaccharide chains. The decrease in degree of methylation in its turn may trigger dierent processes related to texture and rmness. These processes may comprise crosslinking by Ca2 , increasing the hydration at the demethylated sites, enhancing shielding and repulsion forces by the electric charges within the biopolymer matrix of the cell wall as well as decreasing the susceptibility to heat induced b-degradation of pectins and in increasing the susceptibility to polygalacturonase

0260-8774/99/$ see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 0 - 8 7 7 4 ( 9 8 ) 0 0 1 6 0 - 5

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(PG) induced depolymerisation. As such, PE has a long record of confusing and contradictory reports on its eects on observed rmness. In general, PE activity during blanching and processing increases the rmness of the nal (sterilised) product (Bartolome & Ho, 1972; Klein, Hanzon, Irwin, Shalom & Lurie, 1995; McFeeters, Fleming & Thompson, 1985; Pilknik & Voragen, 1991; Wu & Chang, 1990). During storage of fresh fruit and vegetable, however, PE does not seem to aect the rmness (Awad & Young, 1979; Brandy, 1976; Buescher & Furmanski, 1978; Jen & Robinson, 1984; Kanellis, Solomos & Matoo, 1989) or decreases the rmness only to a small extent (Ricard & Noat, 1986a,b). The relation between measured PE activity and observed rmness is inherently complex. One has to consider that the living organism, during its lifetime, is capable of adapting or changing the levels of active enzymes and the accessibility of the substrate. Consequently, the total exerted eect of a measured level of PE activity can never be established based on a single measurement of activity in time. The apparent activity of any enzyme is likely to be the resultant of a complex scheme of biological reactions. The dependence of the apparent activity of PE on different blanching conditions (e.g. temperature) and for batches with dierent maturity at harvest, is likely to be complex as well. Consequently, a simple relation between observed activity and softening is not to be expected. In this paper, a mathematical model is deduced and presented that describes the dynamic changes in PE activity during blanching at dierent temperatures in peaches of cv. Andross. Based on consistent problem decomposition and on consistent application of the fundamental laws of kinetics, a generic model for the eect of blanching on the observed activity of PE in peaches has been developed. The model also accounts for the eect of dierent levels of enzyme activities in batches and for dierent levels of maturity. 2. Material and methods 2.1. Sample preparation and extraction Peaches (cv. Andross) were harvested in July 1994, July 1995 and July 1997 in Northern Greece (Verria) and processed within 24 h after harvesting. The 1997 season samples were harvested at three dierent stages of maturity with a rmness between 32 and 39 N (rm), 18 and 34 N (medium) and 9 and 20 N (ripe) respectively as measured with an Instron Universal Machine tted with an 8 mm plunger traveled (Instron methods used see Tijskens, Rodis, Hertog, Kalantzi & van Dijik, 1998). The halves of 1213 fruits were sliced by hand to 1.5 2 mm thick sections. Approximately 50 g of slices, held

together in a nylon net, were dipped in a 18 l water bath for pre-specied time. The samples were blanched at constant temperatures of 45C, 50C, 55C, 60C, 65C, 70C and 75C in 1994 and 1995 and at 50C, 55C, 60C in 1997. At regular times during blanching, up to 300 s (1994), 600 s (1995) and 900 s (1997), samples were taken at regular intervals and immediately dipped in ice cold water containing Polyclar AT (a water insoluble PVP for binding phenolic impurities. Vendor: SERVA). PE was extracted by homogenising 50 g fresh tissue in the presence of 100 ml ice cold water containing 0.5% suspended Polyclar AT in an Osterizer (a liqueer blender Vendor: Oster, WI, USA) blender for 5 min and stirred for 1 h at 4C. After centrifugation at 10,000 g for 10 min, the sediment was re-suspended in 50 ml icecold 1M NaCl, stirred for 1 h and centrifuged at 10,000 g for 10 min. The pH of the supernatant was adjusted to 7.0 with NaOH and used as such for enzyme analysis. Extractions were conducted in triplicate. 2.2. Assay In 1994 a titrimetric method was used: 25 ml pectin solution (Citrus pectin, DE 75%, Sigma Chem., USA) was incubated in a water-heated vessel at 30o C. The reaction started by adding 1 ml of enzyme solution and was allowed to proceed for 15 min. The amount of 0.01 N NaOH delivered with the 665 Dosimat automatic titrator (Metrohm, Switzerland) was recorded. In 1995, the assay according to Hagerman and Austin (1986) and to Seymour, Preston, Wicker, Lindsay and Marshall (1991) was used. The temperature during the assay was kept at 30C. The results were expressed as nkatal/gFWT: the amount of carboxylic groups in nmoles formed per second per gram of Fresh Weight Tissue. The two methods diered in results for the same activity in a sample. Part of the samples in the 1997 season were analysed with both techniques. The titrimetric technique gave higher activities by a mean factor of 1.8 than the Hagerman and Austin technique. 2.3. Model development The models were developed using a system of problem decomposition (Sloof & Tijskens, 1995). This system is oriented towards underlying processes that cause the observed phenomena, rather than towards the phenomena themselves. The models are based on kinetic mechanisms, assumed and plausible for that particular process, and are developed further by using the general rules of chemical kinetics (Cabral, Best, Boross & Tramter, 1993; Whitaker, 1994; Segel, 1993). The mathematical development was carried out using MAPLEV (Waterloo Maple Software, Waterloo, Ont., Canada), a computer algebra package, capable of

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handling algebraic and dierential equations. Simulations with the dynamic system were carried out by PROSIM (Sierenberg & de Gans, Waddinxveen, the Netherlands), a modelling language, combining the benets of discrete and continuous modelling. 2.4. Statistical analysis Statistical analyses were conducted using the iterative nonlinear regression system of the statistical package GENSTAT (Rothamsted Experimental Station, Rothamsted, UK). No transformations were applied to the data to prevent errors during the estimation (Ross, 1990). The data were analysed as one integral set using time and temperature simultaneously as explaining variables (Tijskens, 1994).

3. Results and modelling 3.1. Experimental data The data from 1994 were measured during a heating period of 300 s of which 8 data points were collected. The data from 1995 were measured during a heating period of 600 s of which 57 data points were collected. As a consequence the number of data points during the rst few minutes of heating is, in the data of 1995, reduced to only 2 or 3 points. This is a substantial reduction of information in the period the most pronounced changes take place. During the trials of 1997, the applied blanching time was extended to 900 s, as only the low temperature range (5060C) was studied.

The raw measured data on PE activity for seasons 1994 and 1995 are shown as symbols in Figs. 1 and 2 respectively. The measured initial PE activity was different for the dierent temperatures, thus hampering interpretation of the results. The data clearly show a decrease in apparent activity in time at dierent constant temperatures. The general shape of the decrease at any temperature appears to be exponential, indicating a rst order denaturation. At higher temperatures the rate with which the activity decreases is faster than at lower temperatures. This expected general behaviour is, however, disturbed at temperatures around 60C. During the 1994 season, blanching at 60C resulted surprisingly in a higher PE activity than blanching at 45C or 50C. During the 1995 season, no dierence was found after blanching at either 55C, 60C or 65C and each temperature treatment resulted in comparable PE activities. As this anomalous behaviour is measured both seasons, a measuring error can be excluded. Some physical or chemical process has to be responsible for this phenomenon, since a decrease of a rate constant with increasing temperature is against all rules and laws of fundamental kinetics and physical chemistry. Another unexpected behaviour can be noticed in the measured data. The activity of PE, remaining after blanching at the dierent temperatures, tends to a lower value at the higher blanching temperatures. Again this should be included in the model development as e.g. an additional reaction process that becomes noticeable at higher temperatures. Although a striking similarity exists in the general behaviour and shape of the activity in seasons 1994 and 1995, especially considering the activity time

Fig. 1. Measured (symbols) and simulated (lines) activity of PE (season 1994) in peaches during blanching at dierent constant temperatures.

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Fig. 2. Measured (symbols) and simulated (lines) activity of PE (season 1995) in peaches during blanching at dierent constant temperatures.

behaviour for each temperature separately, the absolute level of initial activity of PE is somewhat higher in peaches of season 1994 than those of season 1995. This could result from the dierent measuring techniques used in both seasons. The two dierent measuring techniques applied in season 1994 (titrimetric method) and 1995 (colorimetric method) were compared in 1997. The titrimetric method gave readings 1.422.29 (average 1.779, see Table 1) times higher the colorimetric method. This is quite the same as the ratios between the initial activities found in 1994 and 1995 (see Table 2). The fruits from the 1995 season were harvested, as judged according to the colour and hardness of the fruits by the grower, at a somewhat softer and riper stage than fruits from the 1994 season. Apparently the stage of maturity at harvest does not aect the total enzyme activity. These results are conrmed by the results of season 1997, where all three ripening stages showed comparable PE activities at the moment of harvest.

3.2. Enzyme activity and heat denaturation It has been indicated that irreversible denaturation of enzymes by heat behaves according to a rst order reaction mechanism (Cabral et al., 1993; Whitaker, 1994; Wiley, 1994). Reversible denaturation has been indicated to behave according to an equilibrium reaction (Feng, Li & Boersma, 1990). Both systems adequately describe, by a dierent temperature dependence of the constituting reactions, the normally observed enzyme activity, that is an increase in activity at low and medium temperatures followed by a more or less steep decrease in activity at still higher temperatures. First order denaturation was already successfully applied to polygalacturonase activity in peaches during storage (Tijskens et al., 1998), to pectin methyl esterase in potatoes and carrots (Tijskens, Waldron, Ingham & van Dijk, 1997a), to peroxidase activity in peaches, carrots and potatoes (Tijskens, Rodis, Hertog, Waldron, Ingham,

Table 1 Comparing measuring methods at dierent stages of maturity, time and temperature of treatment Time (min) Temperature (C) Maturity stage Activity (nkatal/gFWT) Titration 0 0 9 2 4 9 0 2 4 9 50 50 50 55 55 55 60 60 60 60 Medium Ripe Medium Firm Firm Firm Firm Firm Firm Firm 10.200 9.800 10.000 10.000 15.000 10.833 15.358 15.925 9.583 10.417 Spectrophotometry 5.798 6.712 6.406 5.616 8.146 7.640 5.504 7.487 5.632 7.134 1.759 1.460 1.561 1.781 1.841 1.418 2.790 2.127 1.701 1.460 Ratio

L.M.M. Tijskens et al. / Journal of Food Engineering 39 (1999) 167177 Table 2 Results of statistical analysis (data 1994) based on the classical approach Temperature (C) 45 50 60 65 70 75 kd (s1 ) 0.00951 0.02563 0.00353 0.01020 0.08246 0.06386 PEvar (nkatal/gFWT) 6.620 5.559 7.427 11.12 9.929 10.297 PEfix (nkatal/gFWT) 4.750 4.933 4.412 1.240 1.8 (xed) 1.8 (xed) Nobs 8 8 8 8 3 3 R2 adj 93.8 93.3 94.1 93.7 100 99.9

171

Proxenia & van Dijk, 1997b), to lipase activity in rape seed oil during heat processing (Ponne, Mller, Tijskens, o Bartels & Meijer, 1996) and to lipoxygenase activity in French green beans (Leguijt, unpublished). 3.3. Classical approach 3.3.1. Modelling To obtain some feeling for the data, for the behaviour of the enzyme activity and for the observed anomalies, a `classical' analysis was conducted on the data of 1994 season: for each blanching temperature separately the data were analysed with a simple exponential model: PE PEvar ekd t PEfix Y 1

mains almost constant at 10.512.3 nkatal/gFWT. Simultaneously, the increase in rate of denaturation kd also shows a discrete change but now more between 65C and 70C. 3.4. Integral kinetic approach Based on the results described above, the activity behaviour can be explained by two active congurations of the same enzyme, each with dierent susceptibilities to heat denaturation. In the intermediate temperature region (6065C) a conversion from the bound conguration to the soluble one noticeably occurs. As the exact mechanism of the conversion is not known, the most simple mechanism of rst order reaction for the conversion of the bound into the soluble conguration is applied. This system can be presented by a set of chemical reactions given in Eq. (2). The subscripts refer to the dierent congurations of the enzyme. PEb 3 PEs Y PEb 3 PEna Y PEs 3 PEna Y where the index s indicates the soluble fraction, index b the bound fraction, c represents the conversion reaction and d the denaturation of the enzyme. By applying the fundamental laws of kinetics, this set of chemical reactions can be converted into a set of dierential equations: oPEb kdYb PEb kc PEb Y ot 3 oPEs kdYs PEs kc PEb Y ot where t is the time of heat treatment. At constant temperatures, the solution of this set of dierential equations for the sum of both active congurations of the enzyme (PEtot ) is given in Eq. (4). PEtot PEb PEs PEfix Y 2 3 kdYb kdYs ekdYb kc t kc ekdYs t PEtot PEbY0 kdYb kc kdYs PEsY0 ekdYs t PEfix Y
kdYs kdYb kc

where PE is the total activity, index var refers to the variable part and index x refers to the xed or invariable part of the activity, kd is the rate constant of denaturation at a particular temperature, t the time of blanching. In this scheme PE represents not simply the concentration of enzyme present, but the activity of the enzyme as assessed in the experiments. This is a combination of pure concentration and the specic rate constant, which is the activity exerted to the substrate at assay temperature per mol enzyme. This combination is generally known as the apparent rate of reaction. PEvar represents the concentration of active enzyme that is susceptible to denaturation at that temperature. PEfix represents the concentration of active enzyme that is not susceptible to denaturation and remains active. What the nature is of this observed activity cannot be deduced from these data. Either the enzyme is in a more heat stable conguration or the enzyme is bound to some type of substrate, which makes it more heat stable. It could also be a completely dierent enzyme or a measuring artefact. The heat stable part of PE activity (PEfix ) was not found in potatoes and carrots (Tijskens et al., 1997a). 3.3.2. Statistical analysis The results of the nonlinear regression analyses are shown in Table 2. The xed part of the enzyme activity (PEfix ) roughly shows two dierent levels: at 60C and below, it is about 4.7, above this temperature it is about 1.6 nkatal/gFWT. The total sum of PEvar and PEfix re-

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where index tot represents the total activity of PE, and index 0 indicates the activity initially present. Since the experimental data were gathered as a function of time at dierent but constant temperatures, the analytical solution, given in Eq. (4), can be used for the statistical analysis of the data. As for any chemical reaction, the reaction rate constants kc , kdYb and kdYs depend on temperature. Here, a dependence according to Arrhenius' law (Eq. (5)) is presumed.   ki kiYref e
ii 1 1 ref abs

3.4.1. Statistical analysis Per season. With Eqs. (4) and (5) nonlinear regression was applied to the data of each season separately with blanching time and blanching temperature simultaneously as explaining variables. It can be observed that

the initial level of PE activity is quite variable, especially in the peaches of 1995 (see Figs. 1 and 2). Each temperature series was measured in a single session of one day. It has been noted (K.W. Waldron, IFR, personal communication, Tijskens et al., 1997a) that this variable initial level is common for these methods of determination of PE activity in any product. Consequently, the initial levels have to be estimated separately for each temperature. Some parameters could not be estimated. The conversion of the bound conguration of the enzyme PEb into the soluble conguration PEs is apparently rather complete at higher temperatures. At lower temperatures the enzyme is mainly present in the bound conguration (PEb ). This abrupt change in conguration can be explained by an extremely high energy of activation of the conversion reaction. At the temperatures tested, little information about this conversion and its associated

Table 3 Result of the statistical analysis of PE activity during blanching for each season separate and combined Parameter 1994 Estimate Kinetic parameters kcY ref Ec /R kdYbY ref EdYb /R kdYsY refY 1994 kdYsY refY 1995 EdYs /R Batch parameters PEfixY 1994 PEbY 45Y 1994 PEbY 50Y 1994 PEbY 60Y 1994 PEbY 65Y 1994 PEbY 70Y 1994 PEbY 75Y 1994 PEsY 0Y 1994 PEfixY 1995 PEbY 45Y 1995 PEbY 50Y 1995 PEbY 55Y 1995 PEbY 60Y 1995 PEbY 65Y 1995 PEbY 70Y 1995 PEbY 75Y 1995 PEbY 80Y 1995 PEsY 0Y 1995 Administrative information 60 Tref (C) R2 tot adjY 95.5 R2 1994 adjY R2 1995 adjY N 38 1.2 57,000 0.1352 20,962 1.954 103 41,833 2.071 7.789 6.81 8.60 9.20 8.21 8.63 1.401 s.e. Fixed Fixed 0.0880 4400 3.61 104 4019 0.345 0.856 1.04 0.859 1.02 1.07 1.07 0.900 0 5.598 10.075 8.400 7.364 7.314 6.117 10.454 8.760 0 60 85.4 52 0 0.429 0.818 0.639 0.578 0.596 0.586 0.781 0.925 xed 1995 Estimate 1.2 57,000 0.3459 52,995 4.02 106 50,490 s.e. xed xed 0.0924 3430 4.1 106 6911 1994+1995 Combined Estimate 0.01027 8031 0.008 500 3.910 104 3.330 107 68,143 0 9.200 6.740 10.810 9.940 8.730 9.990 2.240 0 2.370 4.960 4.370 2.720 2.570 0.840 4.700 4.050 4.879 60 91.5 92.8 90.0 90 s.e. 0.00393 2259 Fixed Fixed 1.960 104

5.44 107 10,713 Fixed 1.930 1.860 1.540 1.630 1.570 1.660 1.460 Fixed 1.320 1.240 1.220 1.220 1.190 1.160 1.120 1.160 0.859

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reaction rate constant kcYref is contained in the data. Therefore, in the analyses the parameter kcYref and Ec /R were xed. Preliminary analyses made the xed values as shown in Table 3 plausible. In Table 3 the results for the estimates for seasons 1994 and 1995 are shown. As can be taken from the standard error of estimate (s.e.) the values for the dierent parameters are quite acceptable. The percentage variance accounted for (R2 ) is acceptadj ably high. Seasons combined. As it is very unlikely that the kinetic mechanism of enzyme activation and deactivation will change for the same cultivar over batches and seasons, the kinetic parameters (kcYref , kdYbYref , kdYsYref , Ec /R, EdYb /R, EdYs /R) were estimated in common for both seasons, whereas the initial levels of the activity were estimated separately for each temperature and each season. The kinetic constants for the denaturation of the bound conguration were xed to include a small amount of denaturation, like was found for season 1995 (see Section 4).The explained part was found to be 89% which is quite satisfactory. The parameter kdYsYref could not reliably be estimated in common, and was therefore estimated separately for each season. The results of this analysis are also shown in Table 3. 4. Discussion 4.1. General considerations The results shown, suggest two active congurations of pectin methyl esterase (PE) to exist in peaches. This result is in accordance with the report of Glover and Brady, 1994. At the lower temperature region, the bound conguration (PEb ) prevails, at higher temperatures the soluble conguration (PEs ) prevails by a very rapid temperature dependent conversion. The two congurations of the enzyme have dierent characteristics with respect to heat stability: the reaction rate constant of the denaturation at reference temperature for the soluble conguration (kdYsYref ) is considerably smaller then for the bound conguration (kdYbYref ), which indicates a better heat stability at that reference temperature. The activation energy for the denaturation of the bound conguration (EdYb ) is much smaller (see Table 3, 1994 and combined analysis) than for the soluble conguration (EdYs ), which indicates that the denaturation of the soluble conguration is more aected by a temperature increase then the denaturation of the bound conguration. So, at some higher temperature, the rate constant of denaturation of the soluble conguration will be higher than that of the bound conguration. At that higher temperature, however, all enzyme from the bound conguration will already be completely converted into the soluble conguration. The eect of temperature on the dierent rate constants can be taken from Fig. 3.

Fig. 3. Rates of the dierent reactions (kc , kdYb , kdYs ) in function of temperature.

When the conversion reaction always and consistently precedes denaturation of the bound conguration, the denaturation of the bound conguration will be superuous and undetectable. When the denaturation of the bound conguration consistently precedes conversion, the conversion will be apparently absent. The fact that both reactions are needed fully to explain the observed behaviour, signies that both reactions can precede the other one at some situation in time

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and/or temperature. The reaction rate constants of the dierent reactions and dierent seasons versus temperature are shown in Fig. 3 to make the relative importance of the dierent reaction rate constants more evident. 4.2. Seasonal analysis At lower temperatures the soluble active conguration (PEs ) is much more temperature stable than the bound one (PEb ) as can be taken from the dierence in rate constant of denaturation (kdYbYref and kdYsYref ) and the respective activation energies (EdYb and EdYs ). At these low temperatures, the rate constant of conversion from the bound to the soluble conguration, is small but in the same order of magnitude as the denaturation of the bound conguration. This can be seen as the decrease in activity (see Figs. 1 and 2, lower temperatures). At temperatures above 5055C, however, the rate constant of conversion from the bound to the soluble conguration is always larger than the denaturation of the bound conguration (Fig. 3). So, the bound conguration is rather converted instead of denatured. In Figs. 4 and 5 the results, simulated with the parameter estimates from Table 3, are shown for both seasons. In season 1994 (Fig. 4), a clear increase in activity is observed between 55C and 65C, as can be deduced from the dierent values for the activation energies for the change of PEb by either denaturation (EdYb /R) or conversion (Ec /R) to PEs . Below that region a net decrease in total PE activity

is obtained by denaturation of the bound conguration, above that region by denaturation of the soluble conguration. The net denaturation of the total concentration of PE in 1995 is very small until about 70C (see Fig. 5). The ratio between the concentration of PEb denaturated and the concentration converted to PEs at any temperature equal the inverse ratio of the respective reaction rate constants kdYb and kc . Because of the dierence in activation energies, this ratio depends on temperature. For example at 45C, of the small amount of vanished PEb , 5% is converted to PEs and 95% is denatured. At 60C, PEb activity is almost completely vanished, 85% of which is converted to PEs while only 15% is denaturated. The activation energies of denaturation and conversion (EdYb /R and Ec /R) for the data of 1995 are almost the same (about 55,000). Consequently, the ratio between the change of PEb by either denaturation or conversion to PEs remains basically constant at all temperatures. So, 78% of PEb is always used for the formation of PEs while 22% denaturates. At temperatures above 50C, all PEb is transformed abruptly during the rst seconds of the heat treatment. However, because of denaturation, only 79% becomes available again as PEs . As the denaturation of PEs below 70C is very slow (low kdYs with high EdYs /R), the eect of this process cannot be observed at temperatures below 70C (see Fig. 3). As a result, the total level of PE (PEtot ) stays more or less constant between 50C and 70C. At temperatures above 70C, denaturation of PEs becomes

Fig. 4. 3D simulation of PE activity during blanching at constant temperatures (based on separate analysis season 1994).

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Fig. 5. 3D simulation of PE activity during blanching at constant temperatures (based on separate analysis season 1995).

substantial and results noticeably in a decrease of PEtot . So, the apparently dierent behaviour of the activity in 1995, as compared with 1994, can be explained by the same reaction mechanism, and is therefore more a consequence of dierent parameter values than of a dierent kinetic mechanism.

4.3. Analysis of seasons combined Although some kinetic parameters in the analysis of the seasons combined show quite a large dierence with the values in the seasonal analyses, there is hardly any loss of explaining power as can be taken from the

Fig. 6. 3D simulation of PE activity during blanching at constant temperatures (based on combined analysis 1994).

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equally high explained parts (R2 1994 93% and adjY R2 1995 90% for the combined analysis compared with adjY R2 1994 95% and R2 1994 85% for the separate anadjY adjY alyses). There is a slight decrease in the standard error of estimates (s.e.) for the kinetic parameters. The major advantage, however, of combined analysis is to be found in a tremendous increase in explaining and predicting power for dierent situations and for dierent batches. The fact that the activity of PE in peaches of such different ripeness and harvest dates can be analysed together, with the same model and the most important parameters in common, constitutes a major validation of the principles, assumptions and deduction techniques underlying this model. These results also indicate that modelling should be aimed at explaining and describing the underlying processes in a structural and theoretical way, rather than to describe the measured values as such with some empirical relation. The fact that the soluble denaturation rate constant at reference temperature (kdYsYref ) could not be estimated in common merely signies that the denaturation process is more complex than assumed, and that it does not represent a pure reaction rate constant, but a combination with some entity. The process should be decomposed even further into smaller constituting processes. As this was until now never appreciated, no data are available to conduct such decomposition and analysis. The results of the combined analysis (see Table 3) gave signicantly dierent results only in the kinetic constants of the conversion (kcYref and Ec /R). The most prominent dierence was, however, found in the level of the initial activity of the soluble enzyme (PEsY0 ) which was about one activity unit higher than in the seasonal analyses. Consequently, the level of estimated activity in all other activities is estimated roughly one unit lower. What these small dierences mean for the apparent behaviour can be taken by comparing Fig. 4 with Fig. 6. Although the kinetic constants are not that dierent, the apparent behaviour is drastically changed, especially in the area between 50C and 60C, where too little information is contained in the data. Based on the ndings of Van Impe, 1997 with respect to an innite number of solutions in (dynamic) nonlinear systems, this is not at all surprising. 5. Conclusions The analyses show clearly that data of several seasons may be combined by assigning the seasonal dierences completely to dierent initial concentrations of enzymes and substrates, and by keeping the kinetic characteristics (kref and E/R) constant, without losing any information. This is a strong conrmation of the modelling approach and decomposition strategy used.

The parameters of the models developed can be divided into cultivar specic and batch specic parameters. Getting information and knowledge of the fundamental backgrounds of both the cultivar specic and batch dependant parameters is important both for scientic and for practical purposes. The data sets available are still too small to decide whether the cultivar specic parameters are strictly cultivar specic or species specic. What is the nature of the relation between maturity and PE level is, and what is the exact nature of both congurations is, is still subject of speculation and needs further research. Acknowledgements This study was conducted within the framework of the EU AIR project AIR1-CT92-0278 on The biochemistry and archestructure of fruit and vegetable tissue as quality predictors for optimising storage and processing regimes: Basic research leading to applicable models and rules, partly nanced by the European Union. References
Awad, M., & Young, R. E. (1979). Postharvest variation in cellulase, polygalacturonase, and pectinmethylesterase in avocado (Persea americana Mill, cv. Fuerte) fruits in relation to respiration and ethylene production. Plant Physiolology, 64, 306308. Bartolome, L. G., & Ho, J. H. (1972). Firming of potatoes: Biochemical eects of preheating. Journal of Agricultural and Food Chemistry, 20, 266270. Brandy, C. J. (1976). The pectinesterase of the pulp of the banana fruit. Austral. Journal Plant Physiology, 3, 163172. Buescher, R. W., & Furmanski, R. J. (1978). Role of pectinesterase and polygalacturonase in the formation of wooliness in peaches. Journal of Food Science, 43, 264266. Cabral, J. M. S., Best, D., Boross, L., & Tramter, J. (Eds.). (1993). Applied biocatalysis. Working party on applied biocatalysis of the European Federation of Biotechnology. Chur, Switzerland: Harwood Academic Publishers. Feng, Y., Li, X., & Boersma, L. (1990). The Arrhenius equation as a model for explaining plant responses to temperature and water stresses. Annals of Botany, 66, 237244. Glover, H., & Brady, C. J. (1994). Purication of three pectin esterases from peaches. Phytochemistry, 37, 949955. Hagerman, A. E., & Austin, P. J. (1986). Continuous spectrophotometric assay for plant pectin methyl esterase. Journal of Agricultural and Food Chemistry, 34, 440444. Jen, J. J., & Robinson, M. L. (1984). Pectolytic enzymes in sweet bell peppers (Capsicum annuum L). Journal of Food Science, 49, 1085 1087. Kanellis, A. K., Solomos, T., & Matoo, A. K. (1989). Changes in sugars, enzymatic activities and acid phosphate isoenzyme proles of bananas ripened in air or stored at 2.5% O2 with and without ethylene. Plant Physiolology, 90, 251258. Klein, J., Hanzon, J., Irwin, P., Shalom, N., & Lurie, S. (1995). Pectin esterase activity and pectin methyl esterication in heated Golden Delicious Apples. Phytochemistry, 39, 491494. McFeeters, R. F., Fleming, H. P., & Thompson, R. L. (1985). Pectinesterase activity, pectin methylation, and texture changes

L.M.M. Tijskens et al. / Journal of Food Engineering 39 (1999) 167177 during storage of blanched cucumber slices. Journal of Food Science, 50, 201205. Pilknik, W., & Voragen, A. G. J. (1991). The signicance of endogenous and exogenous pectic enzymes in fruit and vegetable processing. In P. F. Fox (Ed.), Food Enzymology (p. 318). Essex, England: Elsevier Applied Science. Ponne, C. T., Mller, A. C., Tijskens, L. M. M., Bartels, P. V., & o Meijer, M. M. T. (1996). Inuence of microwave and steam heating on lipase activity and microstructure of rapeseed (Brassica napus). Journal of Agricultural and Food Chemistry, 44, 28182824. Rexova-Benkova, L., & Markovic, O. (1976). Pectic enzymes. In R. S.Tipson and D. Horton (Eds.), Advances in Carbohydrate Chemistry and Biochemistry (pp. 323385). Vol. 33. New York, London: Academic Press. Ricard, J., & Noat, G. (1986a). Electrostatic eects and the dynamics of enzyme reactions at the surface of plant cells. 1. A theory of the ionic control of a complex multi-enzyme system. European Journal of Biochemistry, 155, 183190. Ricard, J., & Noat, G. (1986b). Electrostatic eects and the dynamics of enzyme reactions at the surface of plant cells. 2. The role of pectin methyl esterase in the modulation of electrostatic eects in soybean cell walls. European Journal of Biochemistry, 155, 191197. Ross, G. J. S. (1990). Nonlinear Estimation. New York: Springer. Segel, I. H. (1993). Enzyme Kinetics. Behavior and Analysis of rapid Equilibrium and Steady-State Enzyme Systems. New York, USA: Wiley. Seymour, T. A., Preston, J. F., Wicker, L., Lindsay, J. A., & Marshall, M. R. (1991). Purication and properties of pectin esterases of Marsh white grapefruit pulp. Journal of Agricultural and Food Chemistry, 39, 10801085.

177

Sloof, M., & Tijskens, L. M. M. (1995). Problem decomposition: Application in experimental research, statistical analysis and modelling. International Symposium on Intelligent Data Analysis IDA-95, August, Baden-Baden, Germany. Tijskens, L. M. M. (1994). Modelling colour of tomatoes. Advantage of multiple nonlinear regression. Proceedings COST94 Workshop Post-Harvest Treatment of Fruit and Vegetables (p. 175185). 1415 September, Leuven, Belgium. Tijskens, L. M. M., Waldron, K. W., Ng, A., Ingham, L., & van Dijk, C. (1997a). The kinetics of pectin methyl esterase in potatoes and carrots during blanching. Journal of Food Engineering, 34, 371385. Tijskens, L. M. M., Rodis, P. S., Hertog, M. L. A. T .M., Waldron, K. W., Ingham, L., Proxenia, N., & van Dijk, C. (1997b). Activity of peroxidase during blanching of peaches, carrots and potatoes. Journal Of Food Engineering, 34, 355370. Tijskens, L. M. M., Rodis, P. S., Hertog, M. L. A. T. M., Kalantzi, U., & van Dijk, C. (1998). Kinetics of polygalacturonase activity and rmness of peaches during storage. Journal of Food Engineering, 35, 111126. Van Impe, J. (1997). Mathematical concepts and techniques for validation of predictive models. Joint COST 915-COPERNICUS CIPA_CT94_0120 Workshop on Food Quality Modelling, 46 June, Leuven, Belgium. Whitaker, J. R. (1994). Principles of enzymology for the Food Sciences (2nd ed.). New York, USA: Marcel Dekker. Wiley, R.C. (Ed.). (1994). Minimally processed refrigerated Fruits 8 Vegetables. New York, USA: Chapman & Hall. Wu, A., & Chang, W. H. (1990). Inuence of precooking on the rmness and pectic substances of the stem vegetables. International Journal of Food Science and Technology, 25, 558565.