P. Lopez 7 A. Vercet 7 A.C. Sanchez 7 J. Burgos (Y)Laboratorio de Tecnología de los Alimentos, Facultad deVeterinaria, Universidad de Zaragoza, C. Miguel Servet 177,E-50013 Zaragoza, Spaine-mail: jburgos@posta.unizar.es

Z Lebensm Unters Forsch A (1998) 207 :249–252 Q Springer-Verlag 1998

ORIGINAL PAPER

P. Lopez 7 A. Vercet 7 A.C. Sanchez 7 J. Burgos

Inactivation of tomato pectic enzymes by manothermosonication

Received: 23 January 1998 / Revised version: 23 March 1998

Abstract The resistance of tomato pectic enzymes tomanothermosonication (MTS), a combined treatmentof heat and ultrasound under moderate pressure, wasstudied. Pectinmethylesterase (PMF) and polygalactu-ronases (PG) I and II were inactivated much more effi-ciently by MTS than by simple heating. In MTS inacti-vation of these enzymes, the effect of heat and ultra-sonic waves was synergistic. D values [the time requiredfor the (original) enzyme activity to decrease by 90%]for PME heat inactivation at 62.5 7C were reduced 52.9-fold by MTS and those for PG I at 86 7C and PG II at52.5 7C, 85.8-fold and 26.3-fold, respectively.

Key words Tomato 7 Pectinmethylesterase 7Polygalacturonase 7 Manothermosonication

Introduction

Tomatoes are one of the most important vegetablecrops, and in terms of per capita consumption, the lead-ing, processed vegetable. More than 80% tomatoesproduced for processing are consumed in the form oftomato juice and derived products, such as paste,puree, catsup, sauce and salsa [1]. Viscosity is para-mount as a quality attribute in determining the accepta-bility of tomato products to the consumer. Viscosityalso has important economic implications for tomatoprocessors, since higher viscosity reduces the amount oftomato needed in a product to obtain a certain level ofquality.

A tomato product’s viscosity is greatly dependent onthe degree of polymerization of pectic substances. En-zymatic depolymerization of pectin in pulp or serum

causes a great reduction in the viscosity of the product.Pectolytic enzymes liberated during crushing act veryquickly, and should be promptly and thoroughly inacti-vated.

Tomato fruits contain two types of pectolytic en-zymes: polygalacturonase (PG; E.C. 1.2.1.15) and pec-tinmethylesterase (PME; E.C. 3.1.1.11). PG catalyzesthe hydrolytic cleavage of a-1,4-glycosidic bonds ofpectin polygalacturonic acid chains. There are two mainPG isoenzymes in tomatoes [2]. Both are endopolygal-acturonases, but they differ in molecular size and ther-mostability. PG II is composed of a single polypeptidicchain [3, 4]. PG I is composed of two subunits, the po-lypeptidic chain of PG II, which is the catalytic subunit,and the so-called b subunit [2, 5]. PME catalyzes thehydrolysis of the methyl ester bonds of pectin. Al-though there are also different PME isoenzymes in to-mato fruits, their heat resistance seems to be quite sim-ilar [6].

To avoid the viscosity problems associated with thecatalytic activity of pectic enzymes, in the manufactur-ing of tomato juice, tomatoes are rapidly heated at tem-peratures in the range 82–104 7C (“hot break”) imme-diatelly following chopping or crushing to inactivatepectic enzymes. However, heat treatments have also anegative impact on other important characteristics oftomato products, such as color, flavor and nutritionalvalue, which could be avoided by using other enzymeinactivation procedures with little or no heating. One ofthese alternative methods of enzyme inactivation is ma-nothermosonication (MTS) the simultaneous applica-tion of heat and ultrasound under moderate pressure,which has been shown to inactivate a number of en-zymes efficiently [7, 8].

The aim of this work was to explore the MTS resist-ance of tomato pectic enzymes as a first step in investi-gating whether this combined treatment could be an al-ternative to conventional heat treatments of tomatojuice.

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Materials and methods

Enzymes and substrates. PME, PG II and PG I were extractedfrom ripe tomato fruits (cv. Daniela F1 hybrid) and separated bygel filtration chromatography as described by Lopez et al. [6].Fractions used for each experiment contained either PME, PG IIor PG I. Polygalacturonic acid and pectin, DE 60%, (both fromcitrus fruits) were obtained from Sigma (St. Louis, Mo.).

PG assay. PG was assayed by measuring the increase in reducinggroups of the polygalacturonic acid substrate using the methoddescribed by Lever [9]. The reaction was performed at 40 7C for10–60 min and was started by adding 0.25 ml of the PG solution(in 50 mM citrate buffer, pH 4.0, 0.4 M NaCl) to 0.25 ml of a 0.4%aqueous solution of polygalacturonic acid.

PME assay. PME was assayed by acid-base titration. The assaytest consisted of 20 ml of a 0.5% solution of pectin in 0.15 M NaClsolution adjusted to pH 7.0. The reaction took place at room tem-perature and was initiated by adding the PME solution(50–200 ml) to the substrate.

MTS apparatus. MTS treatments were performed in a contin-uous MTS apparatus built in our own laboratory. It consisted of astainless steel cylindrical treatment chamber (diameter, 2 cm;length, 2 cm) to which a sonication horn (model 450; from Bran-son, Danbury, Conn.) was attached. The treatment chamber wassurrounded by a second external chamber used as a refrigerationunit. The temperature of the treatment chamber during the ex-periments was adjusted to the desired value by pumping ice-coolwater through the external chamber at the appropriate flow rateby means of a peristaltic pump (Winter, Munich). The solutionsto be MTS treated were forced through the treatment chamber bya Nemo screw pump (Netzsch Monopumpen, Waldkraiburg, Ger-many). The pressure was maintained at the fixed value by a valvesituated at the end of the treatment circuit. The temperature wasmonitored in the circuit immediately after the valve by means of athermocouple. The treatment time was regulated by adjusting thespeed of the Nemo screw pump to reach a certain (and measured)volumetric rate through the treatment circuit.

MTS treatments. Ultrasonic irradiation was performed at a pres-sure of 200 kPa, an ultrasound amplitude of 117 mM and a fre-quency of 20 kHz. Once the treatment time and temperature hadbeen adjusted, and after passing a volume of the enzyme solution(in 50 mM citrate buffer, pH 4.0, 0.4 M NaCl; volume equivalentto twice the void volume of the treatment circuit) through thetreatment circuit, three samples were recovered at the exit of thetreatment chamber, cooled on ice and immediately assayed forenzyme activity. Residence times in the treatment chamber werecalculated by dividing the chamber volume by the flow rate. Eachtreatment was performed in duplicate.

Enzyme inactivation rates are expressed as D values, the timerequired to for the (original) enzyme activity to decrease by 90%.D values obtained with the MTS treatments are compared withthose obtained with the same preparations by simple heat treat-ments, as published [6].

Results

PME was manothermosonicated at 62.5 7C and 37 7C.As shown in Fig. 1, although heating at 37 7C had nomeasurable effect, in terms of inactivation, on this en-zyme, the MTS treatment at the same temperatureinactivated the tomato PME at higher rates than whenit was heated at 62.5 7C. MTS inactivation is a synergis-tic phenomenon since the rate of inactivation due tothis treatment is much higher than the sum of the rate

Fig. 1 Pectinmethylesterase (PME) inactivation by: simple heattreatment at 62.5 7C (solid line) estimated from experimental dataat 60 7C, 66.4 7C and 70 7C and manothermosonication (MTS) at37 7C (}) and 62.5 7C (L). The dashed line represents the theore-tical additive effect of enzyme inactivation by MTS at 37 7C andheating at 62.5 7C

Fig. 2 Polygalacturonase (PG) I inactivation by: simple heattreatment at 86 7C (G) and MTS at 37 7C (}) and 86 7C (L). Thedashed line represents the theoretical additive effect of enzymeinactivation by MTS at 37 7C and heating at 86 7C

Fig. 3 PG II inactivation by: simple heat treatment at 52.5 7C(solid line) estimated from experimental data for 50 7C, 64 7C and70 7C and MTS at 37 7C (}) and 52.5 7C (L). The dashed line rep-resents the theoretical additive effect of enzyme inactivation byMTS at 37 7C and heating at 52.5 7C

of inactivation by ultrasonic waves at 37 7C and theinactivation rate by heat at 62.5 7C.

PG I and PG II were also manothermosonicated attwo temperatures, 86 7C and 37 7C and 52.5 7C and37 7C, respectively. In the case of PME, the inactivationefficiency of MTS was much higher than that of simpleheating at a temperature of 49 7C (PG I) or 15.5 7Chigher (PG II) (Figs. 2 and 3). For both enzymes, the

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Table 1 D values [time required for the (original) enzyme activi-ty to decrease by 90%] for inactivation of pectolytic enzymes bysimple heating and manothermosonication MTS at different tem-

peratures. D values are means BSE. PME pectinmethylesterase,PG polygalacturonase

Enzyme Treatment conditions, D values and efficiency ratio

MTS Heating Ratio D heat/D MTSat equal temperature

Temp (7C) D (min) Temp (7C) D (min) Temp (7C) D (min) Temp (7C) Ratio

PME 37 4.30B0.9 62.5 0.85B0.1 62.5 45.0B0.1 62.5 52.9PG II 37 2.23B0.3 52.5 1.46B0.05 52.5 38.4B0.1 52.5 26.3PG I 37 3.17B0.2 86 0.24B0.02 86 20.6B0.1 86 85.8

combined effect of heat and ultrasonic waves was sy-nergistic.

MTS inactivation of the three pectic enzymes fol-lows first-order kinetics which allows the expression ofreaction rates in terms of D values. The inactivation ef-ficiencies of heating and MTS under the experimentalconditions described are compared in Table 1, wherethe D values and the MTS efficiency ratio as the D val-ue of the heat treatment divided by the D value of theMTS treatment at the same temperature are present-ed.

Discussion

Results presented here show that MTS has a high inac-tivation efficency with respect to tomato pectic en-zymes. At the lowest temperature explored (37 7C), atwhich neither of the pectic enzymes suffered a measur-able loss of activity within the experimental time-scale(D values estimated from previous work on the sameenzyme preparations [6]: PME 1000 h, PG I2.6!1010 h, PG II 2!104 h), both PG isozymes wereremarkably more MTS sensitive than PME.

Significantly different heat sensitivities have beenreported for the two known PG isozymes of tomato [3,6]. Their sensitivities to MTS at 37 7C are, however,very similar. This is expected, since ultrasonic wavesare known to promote the dissociation of subunits ofpolymeric proteins [10] and PG I only differs fromPG II in having a second thermostabilizing chain (bsubunit).

The existence of various PME isozymes in tomatoeswith different degrees of thermostability has also beenreported [11–13]. Nevertheless, log-plots of residual ac-tivity versus treatment time were linear in the experi-ments described here. Since the only enzyme separa-tion method used was gel permeation, and the PMEisozymes described do not significantly differ in molec-ular weight [14], either Daniela F1 hybrid tomatoeshave a single PME isozyme, or their isozymes have verysimilar heat sensitivity and MTS resistance.

Insufficient data for an accurate calculation of thetemperature dependence of the MTS inactivation pro-

cess was collected, but data describing inactivation at37 7C and 86 7C allowed a rough estimate of the z val-ues. The z values were about 50 7C for PG I and 95 7Cfor PG II, versus 5.6 7C and 9.4 7C, respectively, pre-viously estimated [6] when they were subjected to asimple heat treatment. Although it is not possibleeither to make an accurate estimate of the dependenceof the MTS inactivation rate on temperature for PME,its z value must be about 40 7C (markedly lower thanthat of any of the PGs), compared with 5 7C previouslycalculated [6] when subjected to a simple heat treat-ment. Higher z values for MTS compared to the simpleheat treatment have also been reported for soya beanlipoxigenase [15] and protease and lipase of Pseudo-monas fluorescens [8], although not for horseradish per-oxidase [7]. The different temperature dependence ofthe heat and MTS inactivation reactions suggest that asecond inactivation mechanism, different to that oper-ating in the simple heat treatment, was due to the ultra-sonic waves in the MTS treatment. For lipoxigenase, anFe-containing enzyme, a caged Fenton reaction mecha-nism, implying the production of OH* radicals by watersonolysis, H2O2 formation by radical-radical combina-tion and H2O2 decomposition catalyzed by Fe2c at theactive centre has been proposed [15]. This mechanismcould not operate in either the PG isozymes or PMEunless they were endowed with a transition metal in theactive centre. However, a free-radical mechanism can-not be discounted in the case of PME since it seems tocontain tyrosine and histidine residues in its active cen-tre [16, 17]. These two amino acids are among the mostsensitive to destruction by hydroxyl radicals [18].

With respect to the possible use of the MTS tech-nique for pectic enzyme inactivation, the higher z valueof inactivation by MTS than by the simple heat treat-ment indicates that the former technique is compara-tively more efficient the lower the temperature used.

From the data reported here and previous work it isquite obvious that in order to reduce the activity ofPG I, the most thermostable tomato pectic enzyme, to1% of its original level, it would be necessary to heatthe enzyme at 86 7C for more than 40 min, while thesame effect could be achieved by MTS by heating theenzyme for 30 s at the same temperature. To reach thesame PGI inactivation efficiency during a similar time-

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scale (30 s) by simple heating it would be necessary toraise the temperature to about 97 7C.

The future application of MTS to commercial toma-to juice processing depends on the effects of MTS onother tomato juice characteristics. Experiments to elu-cidate MTS effects on rheological behavior, ascorbicacid content, color, volatiles, and sensory properties arein progress. The use of this technique in industry alsorequires the development of appropriate equipment.

Acknowledgements Thanks are given to the Diputación Generalde Aragón for a scholarship granted to A. Vercet. This work ispart of the project ALI 0584 CP financed by the Spanish Comi-sión Interministerial de Ciencia y Tecnología (CICYT).

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