Reduction of Pectinesterase Activity in a Commercial Enzyme Preparation

Home , Alkaline phosphatase, Pectinesterase

Journal of the Science of Food and Agriculture J Sci Food Agric 85:1613–1621 (2005) DOI: 10.1002/jsfa.2154

Reduction of pectinesterase activity in a commercial enzyme preparation by pulsed electric ﬁelds: comparison of inactivation kinetic models Joaquın´ Giner, Pascal Grouberman, Vicente Gimeno and Olga Martın´ ∗ Department of Food Technology, University of Lleida, CeRTA-UTPV, ETSEA, Avda Alcalde Rovira Roure 191, 25198-Lleida, Spain

Abstract: The inactivation of pectinesterase (PE) in a commercial enzyme preparation (CEP) under high intensity pulsed electric fields (HIPEF) was studied. After desalting and water dilution of the raw CEP, samples were exposed to exponentially decay waveform pulses for up to 463 µs at electric field intensities ranging from 19 to 38 kV cm−1. Pulses were applied in monopolar mode. Experimental data were fitted to a first-order kinetic model as well as to models based on Fermi, Hulsheger¨ or Weibull equations to describe PE inactivation kinetics. Characteristic parameters for each model were calculated. Relationships between some of the parameters and process variables were obtained. The Weibull model yielded the best accuracy factor. The relationship between residual PE and input of electrical energy density was found to be that of exponential decay.  2005 Society of Chemical Industry Keywords: pulsed electric fields; kinetics; pectinesterase; model; inactivation

INTRODUCTION It has become customary to use CEPs in fruit and Pectinesterase (PE; EC 3.1.1.11) is a pectic enzyme vegetable juice technology. Depending on the raw which catalyzes the hydrolysis of the methyl ester materials, final products and process requirements, groups in pectin into low methoxylated pectin and processors use specific enzyme formulations for clar- methanol.1 This enzyme is widespread in higher plants ification, maceration or liquefaction and, afterwards, but also can be found as an extracellular enzyme to achieve increased extraction of profitable compo- 18 generated by microorganisms such as yeasts, bacteria nents, clarity, stability, filterability and yield of juice. 19 and fungi. Most plant PEs show maximal activity at To avoid undesirable effects in food products, and neutral or alkaline pH value but fungal PEs usually to eliminate residual PE after achieving the required have optimal activity at acidic pH value.2 amount of change when CEPs are used, heating 12,20 Pectinesterase controls not only relevant physiolog- is commonly used commercially. Unfortunately, heat treatments also cause unavoidable damage, such ical processes in plants in vivo, such as the control of as cooked flavours, colour changes and losses of vita- cell expansion during cell growth and fruit ripening,3–5 mins and nutrients, that lower the quality of the final but also fruit and vegetable processing. Indeed, PE products. is implicated in textural changes during postharvest Among non-thermal treatments that might be used storage, handling and distribution,6,7 and blanching 8 for reducing activity of enzymes while avoiding the of fruits. PE causes undesirable cloud loss of citrus drawbacks of heating, high intensity pulsed electric 9 juices and gelation of concentrates and may adversely fields (HIPEF) are becoming an alternative method affect viscosity and stability of other food products as to heat treatments. Fundamental aspects, equipment 10,11 well. configurations, applications and other general matters Although PE may cause serious technological and of interest related with this technology have been economic troubles for processing of some food reviewed by authors such as Mart´ın et al,21 Barsotti products, the presence of PE activity is indispensable and coworkers,22,23 Barbosa-Canovas´ et al24,25 and in most commercial enzyme preparations (CEP) which Bendicho et al.26 are applied widely nowadays as processing aids in Inactivation of enzymes by HIPEF has been fruit and vegetable juice production.12– 14 Additional reported by various authors. Castro-Castillo27 reduced applications for PE involve peeling of fruits15 and up to 65% alkaline phosphatase activity in milks. analytical determinations as well.16,17 Vega-Mercado and co-workers reported 90% reduc-

∗ Correspondence to: Olga Martın,´ Department of Food Technology, University of Lleida, CeRTA-UTPV, ETSEA, Avda Alcalde Rovira Roure 191, 25198-Lleida, Spain E-mail: [email protected] Contract/grant sponsor: Comision´ Interministerial de Ciencia y Tecnologıa´ (CICYT), Spain; contract/grant number: ALI97-0774; ALI99-1228 (Received 12 September 2003; revised version received 1 November 2004; accepted 2 December 2004) Published online 18 March 2005  2005 Society of Chemical Industry. J Sci Food Agric 0022–5142/2005/$30.00 1613 JGineret al tion of Plasmin28 and reached 60 or 80% inactivation of protease depending on the medium where the enzyme was dispersed.29 Grahl and Markl¨ 30 observed low reduction of alkaline phosphatase and lactoperoxidase activity under HIPEF. Ho et al31 exposed enzymes in aqueous solution to HIPEF and achieved vast reductions in enzyme activity (70–85%) for lipase, glucose oxidase and α-amylase and moderate reduction (30–40%) for peroxidase and polyphenoloxidase. Inactivation of papain (85%) has been reported by Yeom et al.32 Members of our research group have investigated the effect of HIPEF on polyphenoloxidase,33,34 lipase,35 protease36,37 and polygalacturonase;38 the degree of inactivation for these enzymes ranged from 61.2% (lipase) to 98% (polygalacturonase). Min et al39 observed 80% reduction of lipoxygenase. Figure 1. Effect of temperature on electrical conductivity (σ )inthe In contrast, no inactivation, insignificant loss aqueous solution of the desalted commercial enzyme preparation that of activity or even increase of enzyme activity was used to be HIPEF-treated. under HIPEF have also been reported for alkaline phosphatase,30,31,40 lysozyme, pepsin,31 lactate Switzerland) was used as the PE source. The pH 41 30 40 dehydrogenase, lactoperoxidase, , lipoxygenase, and electrical conductivity (σ )fortherawenzyme 40 peroxidase and polyphenoloxidase. preparation at 20 ◦Cwere4.59and13.27 S m−1, Regarding the effects of HIPEF treatments on PE respectively. The solution with pectinesterase activity activity, there are few published accounts and with to be treated by HIPEF (SP) was obtained by desalting 42 conflicting results. Giner et al have studied tomato and diluting the raw enzyme preparation. These PE and achieved a reduction of its activity up to steps were done to lower electrical conductivity of 43,44 93.8% by HIPEF. Yeom et al have also reported the medium to levels that did not cause troubles a significant degree of reduction (83.2%) of orange such as sparking, excessive heating and dielectric PE activity by HIPEF treatments. However, Van Loey breakdown during HIPEF treatments. Thus, 350 g 40 et al obtained no inactivation for PE from orange. of the raw enzyme preparation were poured into Although research on the effects of HIPEF on an Amicon 8400 stirred cell (Millipore Corporation, enzyme activity has increased in the last decade, it Bedford, MA, USA) and ultrafiltrated through a 5000- is patent that available information is still insufficient Da nominal molecular weight filter (Millipore No and, besides, few approaches of modelling inactivation PBCC07610 poly(ether sulfone) membrane, Millipore of enzymes by HIPEF have been published. Only Corporation, Bedford, MA, USA) until 290 g of 39 our research group and Min et al have applied ultrafiltrate had been obtained; the pressure supplier mathematical models to describe the inactivation of was nitrogen gas (Air Liquid SA, Lleida, Spain) enzymes by HIPEF. at 4.5 bar; ultrafiltration was carried out at room This study has the objectives of evaluating the effects temperature. An equal amount of bidistilled water of HIPEF treatments on a fungal PE in a commercial was then added to the concentrated solution and, enzyme preparation using a bench top system as well after mixing, stored at 4 ◦C for 15 h. Finally, and as to test and compare mathematical models which just before HIPEF treatments, the stored solution were useful to describe the effects observed on PE was diluted in a 1:5 mass ratio with bidistilled water activity from the main process variables involved in at 4 ◦C. This diluted solution constituted the SP, such treatments. which had a pH of 4.60. The effect of temperature on the electrical conductivity of SP is shown in Fig 1. MATERIALS AND METHODS A Testo 240 conductivimeter (Testo GmbH & Co, Reagents Lenzkirch, Germany) was used for measurements of Bromocresol green was purchased from Panreac σ , and a Crison micropH 2000 pH-meter (Crison Qu´ımica, SA (Montcada and Reixac, Barcelona, Instruments, Alella, Barcelona, Spain) was used for Spain), esterified pectin from citrus was from Sigma pH measurements. Chemical Co (St Louis, MO, USA) and sodium hydroxide was supplied by Probus, SA (Badalona, PE activity measurement Barcelona, Spain). All chemicals were of analytical The PE activity was determined spectrophotometri- grade. cally following a procedure based on the methodology by Vilarino˜ et al.2 The procedure consisted in follow- PE source and preparation ing the change in absorbance while an enzyme solution A commercial pectolytic enzyme preparation (Pecti- reacted with the substrate, pectin, in the presence of a nex 100 L, Novo Nordisk Ferment Ltd, Neumatt, dye. For this, 10 µl of enzyme solution was added to

1614 J Sci Food Agric 85:1613–1621 (2005) Reduction of pectinesterase activity by pulsed electric ﬁelds

1990 µl of pectin–dye solution. Enzyme solution for PE activity assays was obtained by diluting 1 volume of SP (either treated or untreated by HIPEF) into 9 vol- umes bidistilled water. The pectin–dye solution was a mixture of 17.9 ml of pectin and sodium hydroxide stock aqueous solution (both 50 g kg−1)and2mlof bromocresol green stock solution (0.17 g kg−1). This solution was adjusted to pH 4.75 by adding drops of 0.1 M NaOH in order to perform PE assays at the optimum pH of the enzyme. This optimum pH had been found in previous experiments. The reactants were poured and mixed into a 1-cm path glass cuvette (Hellma GmbH & Co KG, Mullheim,¨ Germany). The reaction was monitored and recorded at 617 nm in a Phillips Spectrophotometer PU 8700 UV/vis (Cam- bridge, UK). Before pouring into the cuvette and just before PE measurement, pectin–dye solution was warmed at 30 ◦C because preparatory assays showed maximum PE activity in SP at this temperature. The Figure 2. Evolution of temperature versus number of pulses (N)at −1 initial rate of the reaction was computed from the lin- different electric field intensities (E,kVcm ) in the aqueous solution of the desalted commercial enzyme preparation that was used to be ear portion of the curve. One unit of PE activity (UA) HIPEF-treated. was defined as a decrease of 0.001 unit of absorbance per minute and per millilitre of enzyme solution. Ini- tial enzymatic activity of SP (A0)rangedwithinthe interval 340–360 UA. Percentage of residual PE activity (RA) was defined by eqn (1): A RA = 100 t (1) A0 where At and A0 are both arithmetic means of three PE activity measurements which were performed for HIPEF-treated and untreated SP, respectively.

HIPEF treatments The HIPEF treatments were carried out using bench top equipment in a batch process: TG-70 high-voltage Trigger Generator system manufactured by Maxwell Figure 3. Evolution of electrical conductivity (σ ) versus number of Technologies Physics International (San Leandro, pulses at different electric field intensities (E,kVcm−1) in the aqueous CA, USA). This generator stored electric energy in solution of the desalted commercial enzyme preparation that was its 0.1 µF capacitor and supplied exponential decay used to be HIPEF-treated. pulses when discharging the capacitor through the product that was being processed. The treatment (N) at the assayed electric field intensities and is shown chamber consists of two circular stainless steel in Fig 2. Thus, electrical conductivity and number electrodes, 3.4 cm in diameter, assembled in parallel of pulses at the different electrical field intensities plate configuration with a 1.5 cm gap (d); the volume are related as indicated in Fig 3. Temperature was of product in the chamber after whole filling was measured using a Hanna H9043 temperature probe 13.62 ml. The chamber was cooled by air during (Hanna Instruments, Guipuzcoa,´ Spain). HIPEF treatments to avoid too much heating of the Pulse width (τ, s) of an exponentially decay pulse is product in the chamber. stated by eqn (2): Treatments were carried out delivering up to 100 pulses at several peak electric field intensities (E,kVcm−1) in the chamber (19, 25, 30, 33, 36 τ = R · C (2) and 38 kV cm−1). The treatment chamber was filled ◦ with SP at 4 C temperature and then the HIPEF where R() is the load resistance of the product in treatments were run. During these HIPEF treatments, the treatment chamber and C (F) is the capacitance of the maximum temperature reached in the SP (37.2 ◦C) −1 the capacitor. The effective R of the load is given by occurred after 100 pulses at 38 kV cm . The increase eqn (3): of temperature in the SP during HIPEF treatments was d R = (3) recorded after delivering different numbers of pulses A · σ

J Sci Food Agric 85:1613–1621 (2005) 1615 JGineret al

Table 1. Experimental electrical conditions for HIPEF treatments

Peak voltage Electric ﬁeld intensity Input energy density −1 −3 U0 (V) E(kV cm ) per pulse, q(MJ m ) 28.5 19 2.98 37.5 25 4.62 45.0 30 7.30 49.5 33 8.94 54.0 36 10.74 57.0 38 11.90

exponential depletion of enzyme activity; it has already been tested in earlier studies.33– 42 Both Hulsheger’s¨ model (eqn (8)) and the model based on Fermi’s

Figure 4. Evolution of pulse width versus number of pulses (N)at equation (eqn (9)) have been proposed to describe different electric field intensities (E,kVcm−1) in the aqueous solution the inactivation of microorganisms under HIPEF. In of a desalted commercial enzyme preparation that was used to be previous work, these models have been examined42 to HIPEF-treated. describe depletion of PE under HIPEF. If the kinetic behaviour of the enzyme follows a two-parameter where d (m) is the gap between electrodes, A (m2)is form of Weibull’s distribution, RA should be given the electrode area and σ (S m−1) is the conductivity by eqn (10) and the value of the mean to the time to of the product. Thus, substituting R in eqn (2) by the inactivate, tm(µs), which is given by eqn (11), could right-hand side of eqn (3) produces eqn (4), which be used as a measurement of the resistance of the allows computation of τ for each N at each E value. enzyme to HIPEF. Weibull’s distribution has been The results of these calculations are shown in Fig 4. tested to describe surviving microorganisms under HIPEF45 but not yet for enzymes. d · C τ = (4) In all these equations, RA(%) is the percentage A · σ of residual PE activity after a HIPEF treatment time t(µs).RA is the initial residual activity of the Thus, total treatment time (t, s) for a HIPEF treatment 0 enzyme at t = 0(RA0 = 100%), E is the electric was computed as given in eqn (5): − field intensity (kV cm 1), and e is the base of the i=I Napierian logarithms. Further terms are introduced t = τi · Ni i = 1, 2, 3,...,I (5) below following the equations in which they appear. i=1 − · RA = RA · e k1 t (7) where I is the number of intervals taken for computing 0 t and τi is the arithmetic mean of the τ values at the −1 bounds of the ith interval with Ni pulses. k1(µs ) is the first-order inactivation rate constant of Provided that the shape of pulses was an exponential the model. decay, the input of total electric energy density −3 − Q(Jm ) for HIPEF was calculated by eqn (6): − (E Ec) t K RA = RA · (8) U 2 · C 0 t Q = N · 0 (6) c 2 · v t is the extrapolated critical t value for RA equal to where N is the total number of pulses delivered to c 100%, Ec the extrapolated critical value of E for RA the product, U is the peak voltage (V), C is the − 0 equal to 100% and K (kV cm 1) is an independent capacitance of the capacitor (F), and v is the volume constant factor. of the treatment chamber (m3). The input energy supplied per unit of volume in RA each pulse (q) was computed using eqn (6) at N = 1 RA = 0 (9) {E − E N } pulse. Q values at the assayed electric field intensities h( ) + a(N) and peak voltages are shown in Table 1. 1 e

E N is a critical level of E where RA is 50% and Models to describe enzyme inactivation h( ) a(N)isaparameter(kVcm−1) indicating the steepness Experimental data were ﬁtted to the kinetic models of the curve around E N . Both E N and a(N)may stated by eqns (7), (8), (9) or (10) (see below) to h( ) h( ) depend on number of pulses, N. study the evolution of RA in SP under HIPEF treatments. Equation (7) represents the classical ﬁrst- −(t/α)γ order decay kinetic model, which describes an RA = RA0 · e (10)

1616 J Sci Food Agric 85:1613–1621 (2005) Reduction of pectinesterase activity by pulsed electric ﬁelds

α(µs) is the scale parameter and γ is the shape parameter (dimensionless). 1 t = α · 1 + (11) m γ

is the Gamma function. An exponential decay model (eqn (12)) was tried to correlate RA as a function of Q. This model has been assayed for identical purposes.33– 38 Units for 3 −1 exponential factor kq and Q in eqn (12) are m MJ and MJ m−3, respectively.

−kq·Q RA = RA0 · e (12)

Statistical analysis The non-linear regression procedure of the Stat- graphics Plus for Windows 2.1 package (Statistical Graphics Co, Rockville, MD, USA, 1994–1996) was Figure 5. Residual pectinesterase activity (RA) in the aqueous used to fit experimental data to the models. The solution of a desalted commercial enzyme preparation versus total analyses of variance, confidence intervals at p = 0.05 treatment time (t) of HIPEF treatments at different electric field −1 and the additional regression analyses performed to intensities (E,kVcm ). establish relationships between estimated parameters and operation conditions were carried out using the This degree of PE inactivation by HIPEF was within same package. Confidence intervals for all estimated the range of values that Giner et al42 and Yeom et al44 parameters are given with their respective confidence reported for tomato PE and orange PE, respectively, intervals, the product of the standard error of the esti- although with different HIPEF systems and operation mates by Student’s t adjusted for degrees of freedom. conditions from those used in the present work. 46 The accuracy factor Af , suggested by Ross, has been used to analyse the precision of the First-order decay kinetic model tested models. Equation (13) computes Af from J The calculated inactivation rate constant (k1)and experimental observations of RA and their J respective regression parameters for first-order kinetic model at predicted values by the model of concern. the different electric field intensities (E)thatwere assayed are shown in Table 2. The adjusted r2 statistic j=J predicted RAj indicated that this model, as fitted, explained between log( ) observed RA 90.0 and 98.9% of the variability in RA. Values j=1 j of k ranged from 0.81 to 5.6ns−1 and increased Af = 10 J 1 significantly (p = 0.05) with electric field intensity. j = 1, 2, 3,...,J (13) This kind of model has also been assayed by Giner et al42 to model the inhibition of tomato PE For this factor, the nearer the Af value to one, the activity under HIPEF at E values ranging from 5 to − better the accuracy. 24 kV cm 1. However, in that study, the number of pulses (N) was used as independent variable instead of the HIPEF treatment time (t). Rate constants RESULTS AND DISCUSSION

Inactivation of PE activity by HIPEF treatments Table 2. Kinetic rate constant (k1) of the first-order decay kinetic The effects of HIPEF treatments on PE activity in model at different electric field intensities for pectinesterase in the SP at several electric field intensities are shown in aqueous solution of a desalted commercial enzyme preparation that Fig 5. HIPEF treatments were significantly (p = 0.05) was HIPEF-treated effective in reducing PE activity. Since temperatures Electric field intensity Kineticrateconstant that could have caused thermal inactivation of the − E (kV cm 1) k (ns−1) r2 enzyme were never reached, the inactivation that was 1 observed on the enzyme was due to the HIPEF 19 0.81 ± 0.16 0.900 treatments by themselves. The experiments showed 25 2.2 ± 0.6 0.989 that, if either total treatment time or electric field 30 3.9 ± 0.2 0.976 ± intensity increased, PE activities in SP after HIPEF 33 4.4 0.3 0.969 ± treatments were lowered significantly (p = 0.05). A 36 5.1 0.3 0.972 38 5.6 ± 0.4 0.969 HIPEF treatment taking 340 µs total treatment time −1 at 38 kV cm electric field intensity attained 86.8% r2: determination coefficient adjusted for degrees of freedom. maximum inactivation for the enzyme (RA = 13.2%). Significance level, p = 0.05.

J Sci Food Agric 85:1613–1621 (2005) 1617 JGineret al reported by these authors for exponential decay pulses with the first-order kinetic decay model and denotes −3 of 0.02 ms pulse width ranged from 0.75 × 10 to that the k1 –E relationship should in general, follow a 6.4 × 10−3 pulse−1. The conversion of these values non-linear pattern. through the pulse width used in that work implied that k1 for tomato PE inactivation under HIPEF Model based on Weibull’s distribution − − − ranged from 37.5 × 10 3 to 320 × 10 3 ns 1.The The scale parameter (α) and shape parameter (γ ) authors also reported higher k1 values for tomato of the Weibull model were obtained by analyzing PE with increasing electric field intensity. In a first experimental data with eqn (10). The estimates of α approach, comparison of k1 values for both PE and γ , and the determination coefficients adjusted for sources denotes that, under their respective operation degree of freedom (r2) at the assayed E, are listed in conditions, HIPEF treatments reduced the PE in SP Table 3. less effectively than the PE extracted from tomato. The Weibull model exhibited huge capacity to This lower effectiveness is more remarkable in that predict residual PE activity after HIPEF treatments as most HIPEF treatments were performed for PE in SP indicated by the high coefficient obtained (r2 > 0.956) at higher E than in the study for tomato PE. for any of the assayed electric fields. Thus, this model This different behaviour between these sources of is likely to be a useful tool to describe inactivation of PE under HIPEF might be due not only to just dif- enzymes under HIPEF treatments. ferent intrinsic sensitivity between the PEs to HIPEF The scale parameter, which ranged from 764 but also to other important factors, such as medium, to 188 µs, showed a significant (p = 0.05) inverse HIPEF system, treatment chamber, characteristics of dependency on electric field intensity: the higher the electric pulses and electrical conditions. electric field intensity applied, the lower the scale Within the considered interval of E, the results parameter obtained. The shape parameter ranged from of fitting a linear model to describe the relationship 1.11 to 1.71. In contrast to α, there were no significant between k1 and E evinced that this model explained differences (p = 0.05) in shape parameter γ due to 2 = accurately the observed variability in k1(r 0.998). electric field intensity. µ −1 1 Thus, the variation of k1( s ) in terms of E (kV cm ) From α and γ , the values of mean time to inac- can be expressed by eqn (14), which has been written tivation (tm) were calculated according to eqn (11). indicating the confidence intervals for the parameters The values of tm were lowered significantly (p = 0.05) = at p 0.05. when E increased. The depletion of tm(µs) as related to E(kV cm−1) accurately matched (r2 = 0, 979) an expo- =− ± × −3 + ± k1 (4.2 0.5) 10 (266 15) nential decay relationship, which is given by eqn (15): × 10−6 · E (14) (−0.0745±0.015)·E tm = (2767 ± 1325) · e (15) This kind of k1 –E relationship has also been found by Bendicho et al35 when applied to a first-order Because no references on the Weibull equation decay kinetic model to describe the inactivation as a model to describe inactivation of enzymes of lipase after having delivered pulses at E from under HIPEF treatments have been published up 16.4 to 27.4kVcm−1 with the same device as in to now, Weibull parameters for PE have been the current work. However, in studies carried out compared with those reported by Rodrigo et al45 using a laboratory scale pulse device at electric fields for some microorganisms (Lactobacillus plantarum and ranging from 2.2 to 24.3kVcm−1, the exponential Byssochlamys fulva). Experiments in the cited work −1 equations were proposed to relate k1 with E for tomato were carried out at E within 28.7–41.7kVcm . PE,42 apple and pear polyphenoloxidase,33 peach Similarly to PE, for both mentioned microorganisms, polyphenoloxidase34 and polygalacturonase.38 The α and tm decreased as electrical field intensity shape of a k1 –E curve certainly follows a linear pattern increased. In contrast to PE, γ -values reported for and differs intrinsically from the curve resulting from an exponential relationship which considers k1 and E Table 3. Weibull parameters (α, γ , tm) obtained at different electric as independent and dependent variables, respectively. field intensities for pectinesterase in the aqueous solution of desalted Portions of any curve might be well approximate to commercial enzyme preparation that was HIPEF-treated a straight line if values of the independent variable Electric field inten- Scale Shape were compressed within a short interval. This could − sity E (kV cm 1) parameter α(µs) parameter γ r2 also occur within larger intervals far enough from the origin for points belonging to an exponential curve. 19 764 ± 24 1.71 ± 0.8 0.956 Most of the experiments in the present work, as well 25 442 ± 18 1.11 ± 0.11 0.993 as in the study by Bendicho et al,35 were performed at 30 282 ± 13 1.19 ± 0.17 0.984 ± ± higher E zones. Equation (14) should be thoroughly 33 232 15 1.14 0.22 0.973 ± ± restricted within the range of the assayed E to calculate 36 202 16 1.11 0.21 0.973 38 188 ± 16 1.16 ± 0.22 0.974 k1. For instance, if eqn (14) were used for computing −1 k1 at E lower than 15.8kVcm , it would output r2: determination coefficient adjusted for degrees of freedom. = negative values of k1. This fact both is incompatible Significance level, p 0.05.

1618 J Sci Food Agric 85:1613–1621 (2005) Reduction of pectinesterase activity by pulsed electric ﬁelds the microorganisms were lower than unity (γ<1), thus PE and the microorganisms followed different depletion patterns. Besides this, tm values were in a range of 0.71–72 µs for the microorganisms. Therefore, in a ﬁrst approach and according to the Weibull model, PE in SP was shown to be more resistant to HIPEF treatments than the microorganisms.

Hulsheger’s¨ equation The characteristic parameters corresponding to Hulsheger¨ model for PE in SP were obtained from experimental data fitted to eqn (8). Thus, tc = −1 43 ± 4 µs, Ec = 14.8 ± 1.3kVcm and K = 30 ± 4kVcm−1 were the values for critical time, critical electric field intensity and independent constant factor, respectively. Estimates of parameters were significant at p = 0.05 and r2 adjusted for the degree of freedom statistic indicated that this model as fitted explained Figure 6. Effect of the number of pulses (N)onFermimodel parameters. Eh(N): critical electrical field intensity; a(N): steepness 92.4% of the variability in RA. parameter. This model was used to describe inactivation of Compared with the PE studied in this work, Giner pectinesterase in an aqueous solution of a desalted commercial et al42 found that tomato PE began to be inactivated enzyme preparation under HIPEF treatments. The continuous line −1 corresponds to the fit of data to a decaying exponential model, at a lower electric field intensity (Ec = 0.7kVcm ) whereas the broken line hints the average mean of a(N)values. but needing longer exposure to HIPEF (tc = 480 µs). Regarding the independent constant factor, K for tomato PE (39 kV cm−1) did not differ considerably tomato PE.42 However, as a dissimilarity, the effect from K for PE in SP Nevertheless, the smaller K value that N exerted on Eh(N) was more pronounced for obtained for PE in SP indicated a steeper depletion of PE in SP than for tomato PE, provided that the the inactivation of the enzyme and thus less resistance exponential factor in eqn (16) was lower for tomato − to HIPEF treatments than tomato PE. PE (2.4 × 10−3 pulse 1).

Model based on Fermi’s equation Comparison of accuracy of tested kinetic models The critical level of electrical field intensity (Eh(N)) In general, all the tested models showed strong capa- and the steepness parameter (a(N)) were obtained bility (0.900 r2 0.993) to describe the inactivation as a result of fitting experimental data to Fermi’s of PE under HIPEF treatments. Thus, reliable infor- model (eqn (9)). The effect of number of pulses on mation to either fix convenient HIPEF treatments or both parameters is shown in Fig 6. All non-linear predict degree of inactivation of the enzyme at differ- regressions performed to estimate Fermi’s parameters ent electrical conditions may be obtained from each gave r2 values, adjusted for degrees of freedom, of one. Hulsheger¨ constants indicate the minimal condi- 0.923–0.990; thus this model, as fitted, also explained, tions required for beginning inactivation of the enzyme with good agreement, the observed variability in RA. whereas Weibull and Fermi parameters give informa- Steepness parameters were significantly (p = 0.05) tion about how the inactivation occurs. The first-order non-dependent on the number of pulses. So, the set kinetic model can be considered as a simplification of ∼ of a(N) values were conveniently summarized by its Weibull (γ = 1) and Fermi equations (Eh(N) = 0and average mean and standard deviation, which were 9.4 a(N) low enough). This model is a simpler equation and 1.8kVcm−1, respectively. This mean a(N)was for describing the inactivation of the enzyme and the comparable with a(N) values reported for tomato inactivation under PEF might be considered as a first- PE,42 which ranged from 5 to 10 kV cm−1 and from 9.3 order irreversible reaction between the active and the to 5.0kVcm−1, in the case of monopolar and bipolar PEF-inactivated form of the enzyme from a kinetic modes, respectively. However, Eh(N) for PE in SP point of view. declined significantly (p = 0.05) as the number of To contrast predictability among the tested models pulses increased. Under the evaluated conditions, the better, the accuracy factor (Af ) was calculated influence of number of pulses on Eh(N) was properly using eqn (13) considering all assayed experimental (r2 = 0.932) described by the exponential decaying conditions for each model. Provided the Af value relationship given by eqn (16): was nearer to one, this model, based on Weibull distribution, yielded the best accuracy factor (Af = −(0.0089±0.0016)·N Eh(N) = (51.4 ± 1.1) · e (16) 1.064) among the tested models. Conversely, the worst Af value corresponded to Hulsheger¨ equation This kind of relationship between Eh(N) and N (Af = 1.149). Somewhat greater accuracy factors than agreed with the results for these parameters for Af for the Weibull model resulted for the first-order

J Sci Food Agric 85:1613–1621 (2005) 1619 JGineret al

a rule, an increase in the electric ﬁeld intensity, number of pulses, total treatment time and input of electrical density energy yield lower residual PE activity in the HIPEF-treated product. All tested kinetic models show satisfactory accuracy. Because of its high accuracy, the model based on a Weibull distribution is likely to become a usual and practical tool to describe inactivation kinetics of enzymes that have been HIPEF-treated. PE activity as a function of input of electrical density energy decays exponentially.

ACKNOWLEDGEMENTS The authors thank the Comision´ Interministerial de Figure 7. Residual pectin methylesterase activity (RA) in the aqueous solution of a commercial enzyme preparation exposed to HIPEF Ciencia y Tecnolog´ıa (CICYT) of Spain for support treatments at several electric conditions that supplied different input of the work included in projects ALI97-0774 and of electric energy density (Q). The continuous line corresponds to the ALI99-1228 as well as Indulleida, SA (Alguaire, fit of data to a decaying exponential model. Lleida, Spain) for providing the commercial enzyme preparation. kinetic model (Af = 1.073) and the model based on Fermi equation (Af = 1.077). Therefore, the Weibull model showed greater capability to make predictions than the other tested models. This fact might be better REFERENCES explained if the form of inactivation of PE curves 1 Wong DWS, Pectic Enzymes, in Food enzymes: structure and mechanism, ed by Wong DWS. Chapman & Hall, New York, exhibited some lag time, as occurred at low electric pp 212–236 (1995). field intensities, and was just reflected by a higher 2 Vilarino˜ C, del Giorgio JF, Hours RA and Cascone O, Spec- shape parameter. trophotometric method for fungal pectinesterase activity determination. Lebensm-Wiss Technol 26:107–110 (1993). Relationship between PE inactivation and Q 3 Bordenave M and Goldberg R, Purification of pectin methylesterase from mung bean hypocotyl cell walls. Phytochemistry When the input of electrical energy density (Q)was 33:999–1003 (1993). increased, PE activity in SP decreased after HIPEF 4 Schuch W, Improving tomato quality through biotechnology. treatments. This is illustrated in Fig 7. The reduction Food Technol 11:78–83 (1994). of PE in SP could be closely related to the supplied 5 Tieman DM and Handa AK, Reduction in pectin methyles- input of electrical density energy (Q,MJm−3)using terase activity modifies tissue integrity and cation levels in ripening tomato (Lycopersicon esculentum Mill) fruits. Plant a decaying exponential model, which is given by Physiol 108:429–436 (1994). eqn (17): 6 Randhawa JS, Dhillon BS, Bal JS and Bhullar JS, Studies on the pectin methylesterase activity during cold storage of − ± · 3· RA = 100 · e (1.73 0.07) 10 Q (17) Pathernakh pear. J Food Sci Technol 24:71–73 (1987). 7 Barret DM and Gonzalez´ C, Activity of softening enzymes This latter equation was obtained as a result of fitting during cherry maturation. J Food Sci 59:574–577 (1994). the experimental data to eqn (12). The model as fitted 8 Tijskens LMM, Rodis PS, Hertog MLATM, Proxenia N and van Dijk C, Activity of pectin methyl esterase during yield a high coefficient when adjusted for degrees blanching of peaches. J Food Eng 39:167–177 (1999). 2 of freedom (r = 0.964). Tomato PE inactivation 9 Joslyn MA and Pilnik W, Enzymes and enzyme activity, in The by HIPEF treatment as a function of supplied Q42 orange: its biochemistry and physiology,edbySinclairWB. also followed the pattern of eqn (17). However, the University of California, Printing Department, Los Angeles, exponential factor for tomato PE and PE in SP differed CA, pp 373–435 (1961). 10 Bartley DA, Nielsen SS and Nelson PE, Comparison of inver- notably from each other provided that the kp values 3 −1 tase and pectinesterase inactivation in processed tomato pulp. were 68 and 1.73 m GJ , respectively. Thus, PE J Food Quality 17:311–320 (1994). activity in SP was shown to be more resistant to 11 Fayyaz A, Asbi BA, Ghazali HM, Che Man YB and Jinap S, HIPEF treatments than tomato PE. To illustrate and Stability studies of papaya pectinesterase. Food Chem compare both PE activities, it can be noted that, if 53:391–396 (1994). equivalent Q were supplied to 1.73 volume tomato 12 Bauman JW, Application of enzymes in fruit juice technology, in Enzymes in food processing, ed by Birch GG, Blake- PE extract and 68 volume SP, an identical reduction brough N and Parker KF. Applied Science Publishers, Lon- degree of PE activity in both of the products would be don, pp 129–145 (1981). achieved. 13 Speiser W, Enzymes for the fruit juice industry. Fruit Processing 6:487–489 (1996). 14 Dietrich H, Enzymes in fruit juice processing. Fruit Processing 8:105–107 (1998). CONCLUSIONS 15 Rouhana A and Mannheim CH, Optimization of enzymatic Reduction of PE activity in commercial enzyme peeling of grapefruit. Lebensm-Wiss Technol 27:103–107 preparation by HIPEF treatments is achievable. As (1994).

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