Combined thermal and high pressure effect on carrot pectinmethylesterase stability and catalytic activity

Journal of Food Engineering 78 (2007) 755–764 www.elsevier.com/locate/jfoodeng

Combined thermal and high pressure effect on carrot pectinmethylesterase stability and catalytic activity Daniel N. Sila, Chantal Smout, Yusuf Satara, Vu Truong, Ann Van Loey, Marc Hendrickx

*

Laboratory of Food Technology, Department of Food and Microbial Technology, Katholieke Universiteit, Kasteelpark Arenberg 22, B-3001 Heverlee, Belgium Received 19 April 2005; accepted 16 November 2005 Available online 10 January 2006

Abstract Carrot pectinmethylesterase (PME, EC 3.1.1.11) was extracted and purified using affinity chromatography. The effect of pressure and temperature on the stability and catalytic activity of carrot PME was studied for model systems as well as shredded carrots. The purified enzyme was characterized by an estimated molecular weight of 32 kDa and a pI above 9.3. At atmospheric pressure, carrot PME showed an optimal pH of 8.0 at 22.5 °C. The enzyme was rather heat labile (inactivation above 50 °C), however, it showed a notable pressure resistance (up to 600 MPa) especially at low temperatures (<40 °C). The catalytic activity of carrot PME was highly dependent on the temperature and pressure applied. In model and food systems (shredded carrots), optimal PME activity was registered at 50 °C in combination with pressures of about 300–500 MPa. Intact tissues revealed a pronounced PME activity at 60 °C at all pressures studied. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Pectinmethylesterase; Catalytic activity; Enzyme stability; Carrots

1. Introduction One of the most abundant pectinases in plants is pectinmethylesterase (PME, EC 3.1.1.11). PME is a cell wall bound enzyme that catalyzes the demethoxylation of pectins resulting in the formation of carboxylated pectin with release of methanol. Occurrence in bacteria and fungi has also been reported (Rexova-Benkova & Markovic, 1976; Versteeg, 1979). PME belongs to a family of parallel b-helix proteins. The three-dimensional structure of PME in carrots has recently been characterized (Markovicˇ, Cederlund, Griffiths, Lipka, & Jo¨rnall, 2002) and its existence in multiple isozymes (Ly Nguyen et al., 2003; Markovicˇ et al., 2002) which differ in their thermal and pressure stability (Ly Nguyen et al., 2003) has been described. The technological importance of PME is well known (Rombouts & Pilnik, 1978) while its biological significance

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Corresponding author. Tel.: +32 16 321572; fax: +32 16 321960. E-mail address: [email protected] (M. Hendrickx).

0260-8774/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.11.016

is continuously being investigated (Nari, Noat, & Ricard, 1991; Tieman, Harriman, Ramamohan, & Handa, 1992). Stimulation of PME activity before cooking in carrots has been demonstrated to enhance the texture of the final product significantly (Noriko, Teramo, & Michiko, 1997; Roy, Taylor, & Kramer, 2001; Sajjanantakul, Van Buren, & Downing, 1989; Sila, Smout, Truong, & Hendrickx, 2004; Verlinden, 1996; Vu et al., 2004). The demand for carrot based juices and concentrates is increasing (Ly Nguyen et al., 2002) and the extraction yields have been effectively boosted using PME in conjunction with other pectinases (Anastasakis, Lindamood, Chism, & Hansen, 1987; Mutlu, Sarioglu, Demir, Ercan, & Acar, 1999). While PME finds application in the processing of clarified juices, its deleterious effects in cloudy carrot juice and concentrates (Sims, Balaban, & Mathews, 1993), and in other cloudy juices (Cameron, Baker, & Grohmann, 1998; Versteeg, Rombouts, Spaansen, & Pilnik, 1980) cannot be underrated. As a consequence, detailed thermal and high pressure inactivation kinetics of carrot PME have been documented (Balogh, Smout, Ly Nguyen, Van Loey, &

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Hendrickx, 2004; Ly Nguyen et al., 2003; Tijsken, Waldron, Ng, Ingham, & Van Dijk, 1997). However, despite the multiple technological advantages offered by PME, information on the controlled stimulation of PME activity is limited and in some cases lacking. Recently, the application of high pressure has been considered as a powerful tool for modifying enzymatic activity. For some enzymes, low pressures (100–400 MPa) combined with moderate temperatures have been found to enhance enzyme activity significantly (Anese, Nicoli, Dall’Anglio, & Lerici, 1995; Cano, Hernandez, & Ancos, 1997; Van den Broeck, 2000; Verlent, Van Loey, Smout, Duvetter, & Hendrickx, 2004). In this context, this work seeks to investigate the biochemical characteristics of purified carrot PME. The effects of temperature and pressure on the stability and catalytic activity of carrot PME in model and food systems will be endeavored. Such results are the basis for identifying the optimal process conditions for carrot PME activity which can be adopted for industrial applications. 2. Materials and methods 2.1. Materials Fresh carrots (Daucus carota var. Nerac) were bought from a local auction in Belgium. Shredded carrots refer to fresh carrots which were grated into uniform flakes using a grater. Apple pectin (degree of esterification (DE) 70– 75%) was a product of Fluka Chemicals Co. (Buchs SG, Switzerland). All other chemicals were of analytical grade. 2.2. Extraction and purification of carrot PME Extraction and purification of carrot PME was carried out using the method described by Ly Nguyen, Van Loey, Fachin, Verlent, and Hendrickx (2002). NHS (N-hydroxysuccinimide)-activated sepharose (Amersham Biosciences, Uppsala, Sweden) which has a high stability combined with good flow characteristics was used as the coupling matrix (Amersham Biosciences, Uppsala, Sweden). 2.3. Gel electrophoresis A PhastSystem (Amersham Biosciences, Uppsala, Sweden) was used for both SDS-PAGE and IEF experiments. SDS was performed using PhastGel homogeneous 20% and PhastGel Tris-tricine SDS buffer strips. The native low molecular weight SDS standard kit of proteins which ranges from a-lactalbumin (14.5 kDa) to phosphorylase b (94 kDa) (PhastSystems, Amersham Biosciences) was used for estimating molecular weights. Samples were boiled at 100 °C for 5 min in a buffer containing 2.5% SDS and 5% b-mercaptoethanol before application on the gels. The pI was estimated using an IEF standard kit (pH range 3.5–9.3, Amersham Biosciences, Uppsala, Sweden). Gel staining was performed by the silver staining technique according to Heukenshoven and Dernick (1985).

2.4. Enzyme stability study 2.4.1. PME activity assay PME activity was determined by the continuous titration of carboxyl groups formed during pectin hydrolysis using 0.01 M NaOH. An automatic pH-stat (718 STAT titrino, ‘X’ Metrohm, Herisau, Switzerland) was used. Routine assays were performed using 30 ml of 0.4% (w/v) pectin solution (DE 70–75%) containing 0.117 M NaCl at pH 6.5 and 22.5 °C. PME activity in units (U) is defined as the amount of enzyme required to release 1 lmol of carboxyl groups per min under the conditions described above. 2.4.2. Thermal stability of purified carrot PME Thermal inactivation studies of purified carrot PME were carried out in the temperature range of 30–70 °C. Purified carrot PME in 20 mM Bis–Tris buffer (pH 6.5), enclosed in 200 lL glass capillaries (Blau brand, Wertheim, Germany), was heat treated under isothermal conditions for 15 min in a temperature controlled water bath. The samples were then cooled in an ice bath and the residual PME activity was determined titrimetrically at pH 6.5 and at 22.5 °C using 0.4% (w/v) pectin solution (D.E 70– 75%) containing 0.117 M NaCl. The analysis was done in duplicate. 2.4.3. Combined thermal and high pressure stability of purified carrot PME Isothermal-isobaric inactivation experiments were carried out in a multi-vessel high pressure equipment (a six vessel system, Resato, Roden, Netherlands). The equipment can be pressurized up to 1000 MPa in combination with a temperature of 40 to 100 °C. A glycol oil mixture (TR 15, Resato) is used as the pressure medium. The pressure range studied varied from 0.1 to 600 MPa at 20, 40 and 60 °C for 15 min respectively. The temperature in the insulated vessels was first equilibrated at the desired temperature using an external cryostat (Haake F6–C50, Belgium). During the equilibration time, flexible microtubes (0.4 ml Biozym, Landgraaf, The Netherlands) were filled with purified carrot PME. Once the desired temperature was obtained, samples were put into the vessels and the vessels were closed within 3 min. Pressure was built up slowly using a standard pressurization rate of about 100 MPa/min to minimize the temperature rise due to adiabatic heating. After pressure build up, an equilibration period of 3 min was taken into account to allow the temperature and pressure to evolve to the desired value. To account for the effects of adiabatic heating, a blank sample was taken after the equilibration time at each of the respective pressures, cooled in an ice bath and the residual PME activity determined titrimetrically (Ao). The other samples were then pressurized under isothermal-isobaric conditions for exactly 15 min (after pressure build up and equilibration time), cooled in an ice bath and the residual PME activity determined titrimetrically (At). The experiments were done in duplicate.

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2.5. Enzyme catalytic activity studies 2.5.1. PME catalytic activity assay based on methanol formation The PME catalytic activity during thermal or combined thermal and high pressure treatments was determined by measuring the amount of methanol released according to the method of Klavons and Bennett (1986). In this method, methanol is oxidized to formaldehyde by alcohol oxidase (EC 1.1.3.13, Sigma, Belgium) followed by a condensation with 0.02 M 2,4-pentanedione in 2.0 M ammonium acetate and 0.05 M acetic acid to form 3,5-diacetyl-1,4-dihydro2,6-dimethylpyridine. The colored compound produced was measured using an ultraviolet/visible light spectrophotometer (Ultrospec 2100 pro from Amersham Biosciences, Uppsala, Sweden) at 412 nm and 25 °C. 2.5.2. Thermal treatment of purified carrot PME (model system) Pyrex tubes with a screw cap were filled with 1.5 ml of 0.4% pectin solution containing 0.117 M NaCl (blank). A second set of pyrex tubes was filled with 1.5 ml of the enzyme–substrate mixture (30 ml of 0.4% pectin solution containing 0.117 M NaCl and 10–13 units of PME in 20 mM Bis–Tris buffer, pH 6.5) within 3 min. A rack containing the two sets of sample was incubated at the desired temperature for PME activation (20–65 °C) and given an equilibration time of 3 min before the start of the kinetic studies. At preset time intervals, a pair of pyrex tubes (one with a blank and another with the enzyme–substrate mixture) was withdrawn from the water bath and immediately heat quenched at 85 °C for 2 min. The samples were then cooled immediately in an ice bath. The amount of methanol in the samples was quantified as described above. The experiment was done in duplicate. 2.5.3. Combined pressure–temperature treatments of purified carrot PME (model system) The effect of pressure and temperature on purified carrot PME catalytic activity was studied in the temperature range of 30–55 °C and a pressure range of 0.1–600 MPa. The 6-vessel high pressure equipment described above was used. Sample preparation was performed at atmospheric pressure and room temperature before pressurization. Two stock samples, a blank (0.4% pectin solution (pH 6.5 = pH of carrot juice) containing 0.117 M NaCl) and an enzyme–substrate mixture (30 ml of 0.4% pectin solution containing 0.117 M NaCl mixed with 10–13 PME units in 20 mM Bis–Tris buffer, pH 6.5) were prepared. Flexible microtubes (0.4 ml Biozym, Landgraaf, The Netherlands) were filled with each of the samples within 4 min and introduced to the high pressure vessels. The vessels were pre-equilibrated at the desired temperature before loading the samples. After closing the vessels, a slow pressure build up (100 MPa/min) was initiated to minimize the adiabatic rise in temperature. Once the desired pressure was achieved, the individual vessels were

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isolated and finally the central tubing was decompressed. An equilibration time of 3 min was taken into account to allow the temperature to evolve to the desired value. The PME catalytic activity was studied during the isothermalisobaric conditions i.e. after pressure build up and equilibration time. After preset time intervals, each vessel was decompressed and the two samples (blank and enzyme– substrate mixture) were withdrawn. The samples were then heat quenched at 85 °C for 2 min before cooling in an ice bath. The amount of methanol was determined colorimetrically. Samples were analyzed in duplicate. 2.5.4. Preliminary studies for in situ PME activity in shredded carrots To investigate carrot PME activity in situ, a preliminary study was carried out using shredded carrots. Samples were prepared at room temperature by vacuum sealing (up to 11 mbar) 5.0 g of shredded carrots in double film polyethylene packs. The samples were then heat treated at 60 °C for 30 min followed by cooling in an ice bath to avert enhanced enzymatic activity at elevated temperatures. Cooled samples were withdrawn from the polyethylene package and soaked in 5.0 ml of demineralized water (w/v 1:1). At 5 min time intervals, the homogenized mixture was filtered using a millipore filter (MillexÒ-GV, 0.22 lm) to avoid further release of methanol from the carrot tissues and to prevent interference of solids during spectrophotometric analysis. The resulting filtrate was analyzed for the amount of methanol released as a function of time. There was no significant difference (P > 0.1) in the amount of methanol released by increasing the soaking time from 5 to 30 min. Therefore, a soaking time of 15 min was chosen as experimental treatment time. 2.5.5. Combined pressure–temperature treatments of shredded carrots (in situ) Samples were treated in the 6-vessel high pressure equipment described above. Combined thermal and high pressure effects were carried out within a pressure range of 0.1–600 MPa and a temperature range of 30–60 °C. Shredded carrots were vacuum packed up to 11 mbar in weights of 5.0 g using a double film polyethylene pack. The packaged samples were pressurized at a rate of 100 MPa/min up to the desired pressure in thermally equilibrated high pressure vessels for exactly 15 min (after pressure build up and 3 min equilibration time). Once withdrawn from the vessels, the samples were immediately cooled in an ice bath. The samples were then removed from the polyethylene bags and soaked in demineralized water (w/v 1:1) for exactly 15 min. The liquid was then filtered using a Millipore filter (MillexÒ-GV, 0.22 lm) and resulting filtrate analyzed for the amount of methanol released. The effects of pressure build up and equilibrium time during pressurization were also investigated. Carrot samples were prepared as explained above. Immediately after pressure build up and after an equilibration time of 3 min, the samples were withdrawn from the high pressure

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vessel and analyzed for methanol as described above. Two independent treatments each in triplicate were carried out. 2.5.6. Combined pressure–temperature treatments of intact carrots (in situ) High pressure treatments were carried out using a single unit high pressure apparatus (Engineered Pressure Systems International, Temse, Belgium, reactor volume = 590 ml, temperature range = 30 to 100 °C and maximum pressure level = 600 MPa). The pressure transmitting medium was a mixture of propylene and glycol (60% Dowcal, The Dow Chemical Co., Horgen, Switzerland). The pumping system uses an electrically driven high pressure intensifier with a displacement of 0.083 L/min. A fluid flow heat exchange system allows thermostating the system from the outside of the vessel, which was done using 58% ethylene glycol solution (Cryostat Haake N8-KT 50W, Karlsruhe, Germany). The apparatus is fitted with thermocouples (type K) which allow recording of the temperature profile at different levels in the pressure vessel and one pressure sensor (data logger–Cobra 7–10, Mess + technik system GmbH). Before pressurization, the vessel was first thermally equilibrated at the desired temperature (30– 60 °C). At least four carrot cylinders (length = 2 cm, B = 1.2 cm) were heat sealed under vacuum (until 11 mbar) in a double film polyethylene bag and pressurized between 0.1 and 500 MPa for 15 min. The highest adiabatic rise in temperature was about 17 (±2) °C at 500 MPa. After pressurization, samples were immediately cooled in an ice bath. The cold samples were allowed to stand for 15 min in demineralized water (w/v 1:1) before the amount of released methanol was determined. Treatments were carried out in duplicate. 2.5.7. Data analysis The kinetic parameters (Km and Vmax) for PME–pectin interactions, as defined in the Michaelis Mentens kinetic model, were estimated using nonlinear regression analysis (SAS version 5.0, 2001). The heat and pressure stability of carrot PME was investigated by estimating the ratio between the residual enzyme activity (At) and the initial enzyme activity (Ao). A double step approach was used to determine the kinetic parameters of the catalytic activity of purified PME during thermal and high pressure treatments (not for in situ studies because a constant time (15 min) was used). First, the net methanol production due to enzymatic hydrolysis was determined by subtracting the amount of methanol produced in the blank samples from the amount of methanol produced in the enzyme–substrate mixture. Consequently, the initial activity of PME (V0) as described by the slope of the linear part of the curve obtained by plotting the amount of methanol (lg) released as a function of time (min) was estimated. The V0 was then normalized by dividing it by the activity of the enzyme (10–13 PME units) used in the enzyme–substrate mixture as determined titrimetrically at pH 6.5 and 22.5 °C. The normalized value

was reported as PME activity (V1). The second step involved determining the activation energy (Ea) and activation volume (Va) for thermal and high pressure catalytic activities respectively. The temperature dependence of PME activity (V1), as expressed by Ea-values, was estimated using the Arrhenius equation (Eq. (1)) in the temperature domain where PME activity (V1) was increasing (650 °C). This was achieved by plotting the natural logarithm of PME activity (V1) against the reciprocal of the absolute temperature.    Ea 1 1 V 1 ¼ V 1ref exp  ð1Þ R T ref T Similarly, by applying the Eyring equation (Eq. (2)) and by plotting the natural logarithm of PME activity (V1) against the respective pressures, the pressure dependence of PME activity, as expressed by activation volume (Va), was determined.   V a ðP  P ref Þ V 1 ¼ V 1ref exp ð2Þ RT Statistical significance was determined using a one way analysis of variance (ANOVA) method at P < 0.01. 3. Results and discussions 3.1. Extraction and purification of carrot PME Since PME is a cell wall bound enzyme that exists as a complex with pectin through electrostatic interactions (Basak & Ramaswamy, 1996), a high ionic strength solution (0.2 M Tris buffer pH 8 mixed with 1 M NaCl) was used for the extraction. A crude yield of about 4000–6000 units per kg of raw material was obtained from the extractions. By using affinity chromatography, PME was selectively isolated from the crude extract. Most of the unbound compounds were first washed away before loading the crude sample on the column. Loosely bound proteins and phenolics were removed by running phosphate buffer (2 mM KH2PO4, pH 6 containing 0.5 M NaCl) prior to elution with a carbonate buffer (Na2CO3, pH 9.85). Fig. 1a illustrates a successful recovery with two unresolved peaks of PME activity. Interestingly, a similar profile was observed for PME activity in the active fractions (Fig. 1b). As indicated in Fig. 1a, points of inflection in the UV profile corresponded with the points of change in the pH profile. The recovery yield from the crude extract was slightly above 50% after purification. To determine the biochemical characteristics of purified carrot PME, all active fractions were pooled together. By running SDS page, a single protein band with an estimated molecular weight of 32 kDa was observed (Fig. 2a). This is in agreement with literature indicating that purified carrot PME exists as a single isozyme (Alonso, Canet, Howell, & Alique, 2003; Stratilova et al., 1998). However, multiple carrot PME isozymes have been reported by other authors

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Fig. 1. Elution profile of carrot PME illustrating (a) UV profile ( ) and pH profile (–) during the purification of crude carrot PME using affinity chromatography. (b) PME activity in different fractions after purification.

Fig. 2. An electrophoresis diagram of purified carrot PME indicating (a) SDS page for the estimation of the molecular weight and (b) isoelectric focusing for the determination of pI.

0.25

PME activity (ml/min)

(Ly Nguyen, Van Loey, Fachin, Verlent, & Hendrickx, 2002; Ly Nguyen et al., 2003). Isoelectric focusing also revealed a single PME isozyme with a pI greater than 9.3 (Fig. 2b). In fact, most plant PMEs exhibit basic pIs with an exception of a few (Komae, Sone, Kakuta, & Misaki, 1990; Lin, Liu, Chen, & Wang, 1989) unlike most microbial PMEs (Rexova-Benkova & Markovic, 1976). In addition, these results are in agreement with the alkaline form of carrot PME (pI 9.8) described by Markovicˇ et al. (2002).

0.2

0.15

0.1

0.05

0

3.2. Substrate interaction of purified carrot PME Fig. 3 illustrates substrate interaction mechanism of purified carrot PME using apple pectin (70–75% degree of esterification containing 0.117 M NaCl) as substrate. By applying the Michaelis Menten model, a Km value of 0.154 mg/ml and a Vmax of 0.233 ml/min were predicted. Similar results were obtained by Ly Nguyen, Van Loey, Fachin, Verlent, and Hendrickx (2002) for purified carrot PME. Carrot PME shows a high affinity for its substrate.

0

1

2

3

4

5

6

7

Pectin concentration (mg/ml)

Fig. 3. Effect of substrate concentration on purified carrot PME activity. Assay conditions: apple pectin (degree of esterification (D.E.) = 70–75%), 0.117 M NaCl, 22.5 °C, pH 6.5.

The affinity is comparable to that of banana PME (Km – 0.151 mg/ml) and papaya PME (Km – 0.12 mg/ml) (Lourenco & Catutani, 1984; Ly Nguyen, Van Loey, Fachin, Verlent, & Hendrickx, 2002). Contrary, plant

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PMEs with low substrate affinity have also been reported (Giovane, Castaldo, Servillo, Balestrieri, & Quaiuolo, 1990).

Changes in pH alter the state of ionization of charged amino acids and that may influence the substrate binding and/or the catalytic action. Fig. 4 demonstrates the change in activity of purified carrot PME with change of titration pH at 22.5 °C. Changes in pH had a significant effect on the activity of the enzyme. At 22.5 °C, the enzyme was active in the pH range of 5–10, with an optimum at pH 8.0 which lies within the optimum pH range (7–9) for most plant PMEs. Previous studies indicated an optimum pH of about 7.3–7.5 for purified carrot PME (Alonso et al., 2003; Ly Nguyen, Van Loey, Fachin, Verlent, & Hendrickx, 2002; Stratilova et al., 1998). The small discrepancy when compared to literature data can be attributed to the differences in experimental set up, different temperatures for which the optimal pH is reported and different substrate conditions. 3.4. Enzyme stability studies 3.4.1. Thermal stability of purified carrot PME Fig. 5 illustrates the residual activity of purified carrot PME after a heat treatment (30–70 °C) for 15 min. The thermal stability study demonstrates that the enzyme is highly sensitive to heat at temperatures above 50 °C. At 55 °C, almost 50% of the activity of the enzyme was lost, whereas at 60 °C, already 95% of the enzyme was inactivated. This confirms earlier observations that carrot PME is highly thermosensitive as compared to other plant PMEs (Alonso et al., 2003; Ly Nguyen, Van Loey, Fachin, Verlent, & Hendrickx, 2002). 3.4.2. Combined pressure–temperature stability of purified carrot PME The threshold pressures for the inactivation of PME from different sources have been reported to vary from

Residual PME activity

3.3. pH effect on the activity of purified carrot PME

1.2 1 0.8 0.6 0.4 0.2 0 30

40

50 60 Temperature (°C)

70

80

Fig. 5. Thermal stability of purified carrot PME. The residual activity was determined titrimetrically at pH 6.5, 22.5 °C after 15 min treatment at different temperatures.

150 to 1200 MPa, depending on its origin and the medium in which inactivation was performed (Indrawati, Van Loey, Ludikhuze, & Hendrickx, 2001). Fig. 6 illustrates the pressure stability of purified carrot PME from 0.1 to 600 MPa at 20, 40 and 60 °C for 15 min respectively. At low temperatures (640 °C), carrot PME was highly barotolerant within the pressure domain studied. At 60 °C and above 300 MPa, a gradual reduction in PME activity was observed. Kim, Park, Cho, and Park (2001) reported a high decay of carrot PME activity at 60 °C above 400 MPa. Contrary, tomato PME is very resistant to pressure induced inactivation above 600 MPa even at 60 °C (Crelier, Robert, Claude, & Juillerat, 2001; Fachin, 2003). It is important to note that isothermal-isobaric inactivation of purified carrot PME has been reported to reveal an antagonistic effect in the rate of inactivation at low pressures (6300) and high temperature domain (>50 °C) (Ly Nguyen et al., 2003). This antagonistic behaviour has also been reported in other plant PMEs (Fachin, 2003; Ly Nguyen et al., 2002; Van den Broeck, 2000).

0.5

Residual PME activity

1.2

PME activity (ml/min)

0.4

0.3

0.2

1 20 °C

0.8

40 °C 60 °C

0.6 0.4 0.2 0 0

0.1

200

400

600

800

Pressure (MPa)

0 4

5

6

7

8

9

10

11

pH

Fig. 4. Effect of pH on the activity of purified carrot PME measured at 22.5 °C.

Fig. 6. Pressure stability of purified carrot PME. The residual activity was determined titrimetrically at pH 6.5, 22.5 °C after 15 min treatment at different pressure–temperature combinations. To avoid the influence of temperature, the initial enzyme activity was measured at 0.1 MPa after 15 min treatment at 20, 40, 60 °C respectively for each of the pressure stability curves.

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3.5. Enzyme catalytic activity studies

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2.5 0.1MPa

2

200MPa

1.5

ln V 1

300MPa

1

400MPa

0.5

500MPa

0

-0.5 -1 -1.5 0.003

0.00305 0.0031 0.00315 0.0032 0.00325 0.0033 0.00335

1 / Temperature (K) Fig. 7. Temperature dependence of carrot PME catalytic activity at different pressures.

of pressure on the Ea-values. However, shifts on the PME catalytic activity curves with change in pressure were apparent (Fig. 7). In literature, no explanation was found that could account for the variations in Ea-values. 3.5.2. Combined pressure–temperature treatments of shredded carrots (in situ) The effect of pressure (0.1–600 MPa) and temperature (30–60 °C) on in situ PME catalytic activity in shredded carrots is summarized in Fig. 8. PME catalyzed demethoxylation of pectin generally resulted in similar trends in methanol production with change in treatment temperature: an increase in the amount of released methanol with increasing pressure, fol25.00

Methanol (µg/g sample)

3.5.1. Combined pressure–temperature treatment of purified carrot PME (model system) The effect of temperature (20–65 °C) and pressure (0.1– 500 MPa) on the catalytic activity of purified carrot PME is summarized in Table 1. At atmospheric pressure, a gradual increase in the amount of methanol formed was observed with increasing temperature up to 50 °C before declining. From 20 to 50 °C, a 2-fold increase in PME catalytic activity was noted. By further increasing the temperature, the catalytic activity decreased due to the heat inactivation of the enzyme. At elevated pressures, the catalytic activity also increased with increasing temperature up to 50 °C. The most pronounced catalytic activity of carrot PME was observed at 50 °C and 500 MPa. This tendency of enhanced PME activity with increasing pressure has been previously illustrated for tomato PME (Krebbers et al., 2003; Van den Broeck, 2000), orange PME (Cano et al., 1997) and other enzymes such as peroxidase and polyphenol oxidase (Anese et al., 1995). At 55 °C, carrot PME activity was increased with increasing pressure up to an optimum at about 300 MPa. Further increasing pressure resulted in a decreased catalytic activity. The reason for such a change may be related to the gradual inactivation of the enzyme with increasing pressure and temperature. The Eyring equation allowed to describe the pressure sensitivity of the catalytic activity of purified carrot PME adequately (R2 = 0.89–0.99). The activation volumes (Va) are summarized in Table 1. The estimated Va-values were negative and in the range 7.80 to 5.73 cm3/mol. Above 40 °C, increase in temperature clearly resulted in a gradual increase in Va. It should be noted that change in Va is the algebraic sum of volume changes accompanying several events: enzyme–substrate interaction, hydration change of substrate and interacting groups and conformational change of the enzyme upon substrate binding. On the other hand, in the temperature domain where PME activity was accelerated by increasing temperature, the Arrhenius model described the temperature sensitivity of the catalytic activity of purified carrot PME satisfactorily as indicated by the high regression coefficients (R2 = 0.97–0.99). There was no clear trend of the influence

30 °C 40 °C

20.00

45 °C 50 °C

15.00

55 °C 60 °C

10.00

5.00

0.00 0

100

200

300

400

500

600

700

Pressure (MPa)

Fig. 8. Effect of pressure and temperature on the amount of methanol produced in shredded carrots after 15 min treatment times.

Table 1 PME activity (V1) as a function of temperature and pressure MPa

Normalized PME activity (V1) (°C) 20

0.1 200 300 400 500

0.98 ± 0.07a ND ND ND ND

Va (cm3/mol)

ND

30

40

Ea (kJ/mol) 45

50

55

1.08 ± 0.10 1.06 ± 0.11 1.18 ± 0.09 1.74 ± 0.34 0.80 ± 0.21

1.11 ± 0.13 1.60 ± 0.13 2.56 ± 0.27 3.51 ± 0.31 4.74 ± 0.61

1.24 ± 0.12 1.37 ± 0.21 ND 3.80 ± 0.55 4.35 ± 0.43

2.03 ± 0.19 2.25 ± 0.09 5.17 ± 0.28 5.89 ± 0.72 6.01 ± 0.34

1.76 ± 0.09 2.80 ± 0.01 3.25 ± 0.82 2.81 ± 0.57 2.47 ± 0.66

6.29 ± 0.72

7.80 ± 0.70

7.40 ± 1.82

6.98 ± 1.37

5.73 ± 0.56

ND: not determined. a Standard error of regression.

60

65

1.50 ± 0.35 ND ND ND ND

0.54 ± 0.01 ND ND ND ND

ND

ND

48.92 ± 6.92 31.60 ± 0.92 60.12 ± 0.57 47.20 ± 5.98 81.43 ± 6.49

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lowed by a plateau and then a decline. However, at 60 °C, a decline in the amount of released methanol was observed with increasing pressure. This can be explained by PME inactivation at elevated temperature and pressure. The most pronounced PME catalytic activity was observed at 50 °C within the pressure range of 200–400 MPa. This is within the range of the results obtained for purified carrot PME. In literature, pressure induced enhancement of PME activity from freshly squeezed orange juice has been noted (Cano et al., 1997). Similarly, in tomato puree, Krebbers et al. (2003) reported an increase in PME activity with increasing pressure (300–700 MPa) at room temperature. Moreover, Hernandez and Cano (1998) found that pressures of about 335–500 MPa combined with temperatures up to 60 °C reflected an increase in PME activity in tomato puree. This increasing catalytic activity of PME seems to be common among plant PMEs and may be linked to reversible configuration and/or conformation changes of the enzyme and/or substrate (Ogawa, Fukuhisa, Kubo, & Fukumoto, 1990). The effect of pressure build up and equilibration time was also studied within the same temperature and pressure domain (Fig. 9). The amount of methanol released during the dynamic phase varied depending on the applied temperature and pressure. The trend was similar to the one 25.00

Methanol (µg/g sample)

30 °C 40 °C

20.00

50 °C

15.00

55 °C 60 °C

10.00

5.00

0.00 0

100

200

300

400

500

600

700

Pressure (MPa)

Fig. 9. Effect of pressure on the amount of methanol produced in shredded carrots during pressure build up and equilibration time.

observed in the former case and the amounts of methanol released were not significantly different (P > 0.1). This may possibly reflect the short time needed to provoke in situ PME catalytic activity. The instantaneous release of PME from the cell wall and the enhanced contact with its substrate during pressurization may further explain this effect. This might have been enhanced by shredding. To demonstrate the interactive effects of temperature and pressure in the demethoxylation of carrot PME, a response surface plot (Fig. 10a) was modeled. Enhancement of carrot PME activity was observed with increasing temperature and pressure until the optimal conditions. The optimum pressure and temperature were estimated to be about 380 MPa and 50 °C respectively (Fig. 10b). The major advantage of this method is that it includes interactive effects among the variables and therefore, it depicts the net effect of temperature and pressure on PME catalytic activity. 3.5.3. Combined pressure–temperature treatments of intact carrots (in situ) Fig. 11 demonstrates the PME catalytic activity in intact carrots with respect to different pressure (0.1–500 MPa) and temperature (30–60 °C) combinations. The catalytic activity of carrot PME as indicated by methanol production was found to increase with increasing temperature in the pressure range of 0.1–400 MPa. At 60 °C in combination with pressures between 100 and 400 MPa, a pronounced enzyme activity was realized. This is in contrast to the observations made in shredded carrots. A possible explanation for this is the enhanced thermostability and barotorelance of PME in intact tissues as opposed to ruptured tissue. In addition, enzymatic activity has been reported to be strongly dependent on the degree of heat penetration and temperature distribution within tissue (Gonza´lez-Martı´nez, Ahrne´, Gekas, & Sjo¨holm, submitted for publication). In our case, the temperature within the sample equilibrated 5 min after pressure build up which translates to 1/3 of the process time. More so, increased thermal and pressure stability of PME in carrot pieces as compared to carrot juice or purified systems has been reported (Balogh et al., 2004). The rate of texture

Fig. 10. In situ PME activity in shredded carrots as illustrated by a (a) response surface plot and (b) contour plot.

D.N. Sila et al. / Journal of Food Engineering 78 (2007) 755–764

Methanol (µg/g sample)

5.0 4.0 3.0 2.0 30 °C 45 °C

1.0

50 °C 60 °C

0.0 0

100

200

300

400

500

600

Pressure (MPa)

Fig. 11. Effect of pressure and temperature on the amount of methanol produced in intact tissues after 15 min treatment time.

degradation (k-value) and the residual texture after prolonged cooking of carrots has also been shown to be highly dependent on the pretreatment conditions (Sila et al., 2004; Vu et al., 2004) prior to thermal processing. Low temperature blanching (60 °C) is associated with increased methanol production and improved texture in carrots (Anton et al., 2004; Smout, Sila, Truong, Van Loey, & Hendrickx, 2005; Vu et al., 2004). 4. Conclusion Increase in temperature (<55 °C) and pressure (6500 MPa) causes a pronounced stimulation of the catalytic activity of PME in model systems as well as in food systems (shredded carrots). The optimal catalytic conditions for carrot PME activity in model systems are in close range to the optimal catalytic conditions in intact tissues. In intact carrot tissues, a significant increase in PME catalytic activity was noted at 60 °C in combination with pressures of 100–400 MPa. From an industrial point of view, it can be suggested that treatment conditions that increase the demethoxylation of pectin such as temperature and pressure may be interesting for the optimal utilization of endogenous carrot PME. This is in line with previous work (Sila, Smout, Truong, Van Loey, & Hendrickx, 2005) on the texture degradation of carrots which indicated that it is possible to design treatments that maximize PME activity thus leading to tissue firming in fruits and vegetables by modifying the pectin structure. Acknowledgement The authors acknowledge the Research Council and the Interfaculty Board for Development Cooperation of K.U. Leuven as well as the Fund for Scientific Research, Flanders (FWO) for financially supporting this research. The input of all co-workers of the Laboratory of Food Technology is highly appreciated. References Alonso, J., Canet, W., Howell, N., & Alique, R. (2003). Purification and characterization of carrot (Daucus carota L) pectinesterase. Journal of the Science of Food and Agriculture, 83, 1600–1606.

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