Reduction of Pectinesterase Activity in a Commercial Enzyme Preparation

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.

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