Xylanase B from the hyperthermophile Thermotoga maritima as an indicator for temperature gradients in high pressure high temperature processing

Innovative Food Science and Emerging Technologies 12 (2011) 187–196

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Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t

Xylanase B from the hyperthermophile Thermotoga maritima as an indicator for temperature gradients in high pressure high temperature processing Liesbeth Vervoort a, Iesel Van der Plancken a, Tara Grauwet a, Priscilla Verjans b, Christophe M. Courtin b, Marc E. Hendrickx a, Ann Van Loey a,⁎ a Laboratory of Food Technology, Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems (M²S), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22 box 2457, B-3001 Heverlee, Belgium b Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Center (LFoRCe), Department of Microbial and Molecular Systems (M²S), Katholieke Universiteit Leuven, Kasteelpark Arenberg 22 box 2463, B-3001 Heverlee, Belgium

a r t i c l e

i n f o

Article history: Received 17 September 2010 Accepted 12 January 2011 Editor Proof Receive Date 10 February 2011 Keywords: Pressure–temperature–time indicator (pTTI) High pressure high temperature (HPHT) processing High pressure sterilization Temperature uniformity Xylanase Thermotoga maritima

a b s t r a c t Within the scope of high pressure food sterilization, an important issue that should be taken into account in refining process and equipment design is the time- and position-dependent temperature gradient that exists throughout the pressure vessel and the product load. Since enzymes from thermophilic microorganisms show good prospects for the development of indicators to map out the temperature non-uniformity in high pressure high temperature (HPHT) processing, in this work, the potential of xylanase B from Thermotoga maritima (XTMB) was investigated. Its inactivation at isothermal–isobaric conditions was best described by a first-order model. The pressure dependence of the D values was negligible at HPHT, the temperature dependence however was substantial. The Thermal Death Time (TDT) model, and its corresponding parameters, describing this large temperature dependence were successfully validated under dynamic processing conditions, relevant for industrial HPHT applications. Industrial relevance: Despite extensive research progress on high pressure high temperature (HPHT) processing as a new food sterilization technique, food industry should be aware of a possible non-uniform temperature distribution, occurring in the pressure vessel and its consequence for the quality and safety of treated products. Since direct measurement of the temperature distribution is not feasible with the measuring devices currently available and constructive computation of the temperature profile by numerical simulation is inadequate, the development of specific temperature-sensitive wireless sensors, or pressure–temperature– time indicators (pTTIs) can be put forward. In this work, xylanase B from Thermotoga maritima (XTMB) was evaluated as a potential enzymatic indicator for mapping the temperature non-uniformity in HPHT processing. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction In recent decades, high pressure (HP) processing has emerged as an industrially adopted method for food pasteurization. This technology has demonstrated its capability of delivering a wide range of high quality chilled products with extended shelf life, such as rare and cooked meats, fish and seafood, dairy and vegetable products and ready-to-eat meals. HP technology, as commercially applied today, typically uses conditions of 400 to 600 MPa at ambient temperature, which only inactivate vegetative microorganisms, not spores, and consequently is unable to produce low-acid shelf-stable products. The ability to produce low-acid pressure-treated products, with an equivalent ⁎ Corresponding author. Tel.: + 32 16 32 15 72; fax: +32 16 32 19 60. E-mail address: [email protected] (A. Van Loey). URL: http://www.biw.kuleuven.be/lmt/vdt (A. Van Loey). 1466-8564/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2011.01.006

microbiological safety as foods subjected to thermal sterilization, by pressure alone has not yet been achieved (Smelt, 1998; Heinz & Knorr, 2005; Black et al., 2007). Various authors have proposed HP to be combined with an additional factor, mostly high temperature (HT), to increase its effectiveness in view of food sterilization (hurdle concept) (Hoover et al., 1989; Wilson & Baker, 1997; Meyer et al., 2000; Wilson et al., 2008). Commercial in-pack thermal sterilization involves extensive thermal treatment because of slow heat penetration to the core of the product and subsequent slow cooling. These thermal processes induce undesirable quality changes, such as off-flavor formation, texture softening, change in color and loss of vitamins. In fact, a number of foods exist that cannot be converted into shelf-stable products by means of retort processing because of the non-acceptable or low quality values obtained after long exposure to heat (BarbosaCánovas & Juliano, 2008). In this case, HPHT processing can be an alternative solution. One benefit of generating high pressure is that it causes a temperature increase as a result of compression heating.

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Although this increase was originally considered irrelevant in HP studies, later research demonstrated that the thermal effect in HP processing is not to be disregarded and does significantly contribute to microbial and enzyme inactivation (Hendrickx et al., 1998; Otero et al., 2000). When taking advantage of compression heating in an ingenious way, higher product quality and lower energy consumption can be achieved (de Heij et al., 2003). A single pressure pulse of 600– 900 MPa will increase the temperature of an adequately preheated sample from 60–90 °C to 100–125 °C in less than a minute. After a holding period of about five minutes or less, also a fast cooling is obtained at pressure release (Wilson & Baker, 1997; van Schepdael et al., 2002; de Heij et al., 2003; de Heij et al., 2005). This instantaneous volumetric heating and cooling results in shorter processing times compared to conventional thermal processing, which is impaired by slow thermal conduction into the center of the can or pack. As a consequence, food products with better retention of color, flavor, texture and nutrients can be obtained (Krebbers, Matser, Koets, Bartels, et al., 2002; Krebbers, Matser, Koets & van den Berg, 2002; Matser et al., 2004; Rodrigo et al., 2007; De Roeck et al., 2008; Leadley et al., 2008; De Roeck et al., 2009). Recent publications claim that the shorter processing time is the key benefit of HPHT treatment over retort sterilization (Matser et al., 2004; de Heij et al., 2005; Barbosa-Cánovas & Juliano, 2008); therefore this technique is also often referred to as pressure-assisted thermal sterilization (PATS). Moreover, this technology was recently accepted as a new food sterilization process by the U.S. Food and Drug Administration (FDA) (Bricher, 2009). High pressure is considered to be instantaneously and uniformly transmitted to all points within the HP vessel (Pascal principle) and therefore, regardless of the product size, each product (fraction) is exposed to an identical stress level. However, this statement does not hold for the temperature distribution. The extent of compression heating is not only dependent on the pressure change, but also on the initial temperature, thermal expansion coefficient, density and thermal capacity of the material pressurized. The content of a filled HP vessel is a heterogeneous system consisting of product, packaging material and pressure-transmitting fluid. As a result, different temperature increases will arise during pressure build-up, despite a potentially uniform initial temperature distribution. In addition, the steel vessel wall will not heat up by compression. Subsequently, heat transfer between the product, pressure medium, vessel wall and surrounding environment will occur. Since small differences in temperature can have significant effects on microbial and enzyme inactivations, these time- and position-dependent temperature gradients result in an inhomogeneous process impact, which concerns the quality and safety of the product (Denys et al., 2000; Hartmann & Delgado, 2002). Therefore, simulation or detection of the temperature distribution as a function of time and position is indispensable for process design and validation of HPHT processes. Direct measurement of the temperature distribution throughout the HP vessel and in-pack is not feasible with the measuring devices currently available and entails some practical problems, as for instance the placement of thermocouples, perturbation of heat transfer, sealing of thermocouple passages, etc. Furthermore, constructive computation of the temperature profile by numerical simulation requires accurate values of the pressure- and temperature-dependent thermal and physical properties of the relevant materials for the applied heat and mass transfer equations. In case of water, databases of its thermophysical properties under pressure exist (US National Institute of Standards and Technology, NIST, and International Association for the Properties of Water and Steam, IAPWS), whereas for other food (model) systems these data are quite sparse (Denys et al., 2000; Otero et al., 2000; Rasanayagam et al., 2003; Patazca et al., 2007). In addition, such models need to be validated. To circumvent these problems, the development of specific temperature-sensitive wireless sensors, or pressure–temperature–

time indicators (pTTIs), can offer a solution (Van der Plancken et al., 2008). To date, some research has been performed on the development of different types of pTTIs in the pressure–temperature window of HP pasteurization (Ludikhuyze et al., 1997; Minerich & Labuza, 2003; Claeys et al., 2003; Bauer & Knorr, 2005; Grauwet et al., 2009; Grauwet et al., 2010a; Grauwet et al., 2010c). However, for HP sterilization, no publications on a successfully developed indicator are available so far. Grauwet et al. (2010b) developed a first prototype of a protein-based pTTI for HPHT applications, yet for HP sterilization the indicator had some drawbacks: (i) the thermal stability at atmospheric pressure was rather limited (5 min at 90 °C would reduce the activity to 38%), (ii) the application window was restricted to mild HPHT processing conditions (90–110 °C, 400–700 MPa, 0–20 min), at which little spore inactivation is achieved, and (iii) the temperature sensitivity at HPHT was rather limited (zT = 22.6 ± 1.3 °C, recalculated from the data). In order to be applicable in the pressure–temperature window of HP sterilization, a possible candidate sensor has to meet a number of requirements: 1) The indicator must be user-friendly with an accurate and reproducible read-out of an irreversible response. 2) It should be heat-stable at atmospheric pressure. On application, the indicator will be inserted in a HP vessel, already set at a relatively high temperature and thus, no significant loss in activity should occur before pressure build-up. 3) Changes in response must be temperature-sensitive at HPHT combinations in order to be able to map out the temperature distribution in the vessel. In this context, enzymes display a number of favorable characteristics: (i) their inactivation at elevated pressure and temperature is pressure- and/or temperature-dependent and can be used as a sensor response, (ii) for many enzymes, there are easy activity assays available that can accurately and precisely determine the level of residual activity, and (iii) their inactivation kinetics can be manipulated in several ways (e.g. by solvent engineering, as shown by Grauwet et al., 2010c) in order to obtain the desired pressure and temperature sensitivity. Enzymes originating from thermophilic microorganisms form an interesting pool for potential candidate indicators to choose from. Not only do they already meet the second requirement of a candidate sensor, in most cases they are also pressure-stable (Eichler, 2001; Mombelli et al., 2002). In fact, thermophilic proteins are often even stabilized by pressure, whereas their mesophilic counterparts are inactivated (Hei & Clark, 1994; Mombelli et al., 2002). In this work, the potential application of xylanase B from Thermotoga maritima (XTMB) as an enzymatic sensor in the processing window of HP sterilization was investigated. This enzyme has proven to be quite heat-stable (Simpson et al., 1991; Winterhalter & Liebl, 1995; Sunna et al., 1997; Jiang et al., 2001; Ihsanawati et al., 2005) and its use in several industrial applications such as paper pulp bleaching and improvement of the bread making quality is discussed by various researchers (Chen et al., 1997; Vieille & Zeikus, 2001; Jiang et al., 2004; Jiang et al., 2005). The three requirements for a candidate indicator for HPHT processing mentioned above were verified step by step. When these conditions are fulfilled, the next step is a kinetic calibration of the enzyme inactivation at isothermal–isobaric conditions. In a final stage, the obtained kinetic model and its corresponding parameters must be validated under dynamic processing conditions for the indicator to be applicable in industrial HPHT treatments. 2. Materials and methods 2.1. Solution of XTMB The enzyme, XTMB, was recombinantly produced in Escherichia coli and purified from the cell lysate through chromatographic

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techniques as described by Dornez et al. (in press). SDS-PAGE showed only a single band, indicating a high purity level (results not shown). A stock solution of 0.19 g/l enzyme in 0.1 M MES (2-(N-morpholino) ethanesulfonic acid)–NaOH buffer pH 6.5 was stored at −80 °C. Since the pKa of MES buffer has a high temperature dependence (Dawson et al., 1969), the stock solution was diluted in acetate buffer, a temperature-stable buffer, before thermal and HPHT treatment, to avoid pH effects on the temperature sensitivity of the candidate sensor. A treatment solution was prepared by a 75-fold dilution in 0.2 M sodium acetate buffer pH 5.0 with various concentrations of urea (cfr. 3.1.3). 2.2. Sample treatment 2.2.1. Isothermal high temperature treatment In order to study the temperature sensitivity of XTMB at ambient pressure, isothermal treatments were conducted in a temperaturecontrolled oil bath (Thermo Haake, The Netherlands) within a temperature range of 80–110 °C. To ensure immediate heat transfer during heating and cooling, glass capillaries (150 × 1.55 mm, 1.15 mm inner diameter, Hirschmann laborgeräte, Germany) were used as a recipient. At predetermined treatment times, the samples were immediately transferred to an ice bath to stop further denaturation. At each temperature time combination a single treatment was performed. 2.2.2. High pressure high temperature treatment All HPHT experiments were performed in a laboratory-scale, multi-vessel HP unit (custom-made, Resato, The Netherlands). The six individual vessels (43 mL, 20 mm internal diameter) are surrounded by a heating coil, connected to a thermostat, and by an isolating outer layer. This system allows computer-controlled pressure build-up, up to 800 MPa and temperature control, up to 120 °C. Pressure inside the vessels and temperature in the center of the vessels were logged by a pressure sensor and thermocouples attached to the vessel stoppers respectively. The pressure medium utilized was propylene glycol (PG fluid, Resato, The Netherlands). The treatment solution of XTMB was divided over flexible microcentrifuge tubes (250 μL, 3 × 0.5 cm, polypropylene, Carl Roth, Germany), hermetically sealed and retained on ice before treatment. Two types of HPHT experiments were carried out: (i) experiments under isothermal–isobaric conditions and (ii) experiments under dynamic conditions. After pressure release, the samples were transferred to an ice bath to stop further denaturation. Each pressure temperature time combination was performed only once.

189

was determined experimentally in advance for each HPHT combination under consideration, using thermocouples (type J, 36.8 mm, attached to the pressure vessel stopper) inserted in a sample holder with a small hole at one side, filled up with water. When the desired initial temperature was reached, pressure was built up at a high rate (from 0.1 to 150 MPa in 5 s and from 150 MPa to the set pressure at 10 MPa/s) to minimize the dissipation of compression heat and the duration of the dynamic phase of the process. The temperature increase due to compression is larger in the thin layer of pressure medium around the sample holders, than in the water and the sample inside, because of differences in initial temperature and compression heating. Nevertheless, fast dissipation of this heat to the vessel wall and to a lesser extent to the sample holder is assumed. When the desired pressure was attained, the individual vessels were sealed and the isolating properties of the sample holders, plus the set temperature of the vessel walls, ensured further isothermal conditions after an equilibration period of 90 s. The sample taken at this point was considered as that of zero treatment time. After various preset holding times at constant pressure and temperature (which are referred to as the actual treatment times (tiso)), the individual vessels were decompressed. Each sample was transferred to an ice bath in exactly 1 min after pressure release. The inactivation of XTMB at isothermal–isobaric conditions was investigated at process temperatures between 100 and 115 °C, and pressures between 500 and 700 MPa. 2.2.2.2. Dynamic conditions. In contrast to the above, during HPHT experiments at dynamic conditions, no sample holders were used, but samples were treated directly in the pressure medium. In consequence of compression heat loss to the surroundings of the pressure vessel, dynamic temperature profiles were obtained. An example of the pressure and temperature profiles measured during a dynamic HPHT treatment is depicted in Fig. 1 (right). When the pressure vessels, with stoppers, and the containing pressure medium attained the desired initial temperature, the samples were fixed to a thermocouple, attached to a vessel stopper, and the vessels were closed again. In this way, the temperature profile is registered at the exact position of the sample tube. 1 min after insertion of the samples, pressure was built up at various build-up rates. When the intended pressure was reached, the vessels were all sealed and after different preset holding times, the individual vessels were decompressed. Exactly 1 min after pressure release, the samples were transferred to an ice bath. For these treatments, initial temperatures between 80 and 95 °C, and pressures between 500 and 700 MPa were applied. 2.3. Activity measurement

2.2.2.1. Isothermal–isobaric conditions. To acquire isothermal–isobaric conditions at HPHT, a new protocol for performing HPHT experiments was followed. An example of the pressure and temperature profiles measured during a HPHT treatment at isothermal–isobaric conditions is depicted in Fig. 1(left). The aim of the strategy is to make use of the compression heat to attain a specific high temperature and to further maintain this temperature at a constant level. To this end, the sample tubes were inserted in specially designed, cylindrical, polyoxymethylene acetale polymer (POM) sample holders (85 mm length, 12 mm inner diameter, and 3 mm wall thickness) with a movable stopper and filled up with water. These sample holders were made to size to fill the pressure vessels as complete as possible, so the ratio of sample holder volume to pressure medium volume is large and constant. All sample holders (with samples) were pre-equilibrated at 10 °C in a cryostat. Next, the sample holders were transferred to the pressure vessels, already equilibrated at the desired process temperature. The vessels were closed and, by means of heat transfer from the pressure medium to the samples, the temperature in the sample holders was allowed to rise to a certain initial temperature, required to obtain the desired process temperature after pressure build-up. This initial temperature

2.3.1. Principle The residual XTMB activity was determined by means of an optimized colorimetric assay. The substrate employed was a ready-touse solution of partially depolymerized wheat arabinoxylan with groups of Remazolbrilliant Blue dye (K-AZOWAX 09/04, Megazyme, Ireland). On incubation of this substrate with an endo-xylanase, the arabinoxylan is depolymerized by an endo-mechanism to produce low-molecular-weight dyed fragments which remain in solution after addition of ethanol. The remaining high-molecular-weight material can be removed by centrifugation and the increase in absorbance at 590 nm of the supernatant can be related directly to the enzyme activity. 2.3.2. Procedure In order to perform measurements within the linear range of the absorbance versus enzyme concentration, an 8-fold dilution of the treated samples was prepared in 0.1 M MES buffer pH 6.5 containing 0.5 g/l bovine serum albumin (BSA) (cfr. 3.1.1). The enzyme solution was first allowed to equilibrate at 90 °C for 10 min after which the

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120

800 700

700

100

600

100

600

300

80

500

60

400 300

40

200

T (°C)

60

400

p (MPa)

80

500

T (°C)

p (MPa)

120

800

40

200 20

100

0

0 -4

-2

0

2

4

6

20

100 0

0 0

8

1

tiso (min)

2

3

4

5

6

7

tabsolute (min)

Fig. 1. Example of pressure (− −) and temperature (—) profiles measured during HPHT treatments. Left: HPHT treatment under isothermal–isobaric conditions at 700 MPa and 115 °C, with tiso the time at isothermal–isobaric conditions, which is referred to as the actual treatment time. Right: HPHT treatment under dynamic conditions at 700 MPa and an initial temperature of 80 °C.

substrate solution was added. The subsequent hydrolysis reaction was terminated after 20 min by addition of ethanol and transfer of the mixture to an ice bath. The precipitated high-molecular-weight, nondepolymerized substrate was removed by centrifugation (15 min at 2655 × g and 4 °C) and the absorbance of the supernatant was determined at 590 nm (25 °C). One arbitrary unit of xylanase activity was defined as the amount of enzyme required to increase the absorbance with 0.001 units in one minute under the defined test conditions. As a reaction blank, the buffer that was used for preparing the treatment solution, was employed, instead of a treated sample. The residual activity after treatment was calculated as the ratio of the activity of the untreated sample i.e. A/Au. The activity in each sample was measured in duplicate and averaged. 2.4. Kinetic data analysis 2.4.1. Kinetic modeling and parameter estimation for enzyme inactivation under isothermal–isobaric conditions A two-step regression procedure was performed to analyze the kinetic inactivation data obtained after (HP)HT treatment. First, the decrease in activity as a function of treatment time was described by a first-order model: dA = −kA dt

ð1Þ

where A is the enzymatic activity at time t (min) and k the inactivation rate constant (min− 1). A commonly used concept in pharmaceutical and food technology (in the case of first-order reactions) is the Thermal Death Time (TDT) model. The decimal reduction time D is defined as the time, at a given temperature and pressure, needed for a 90% reduction of the initial activity and is directly related to k: D=

ln ð10Þ : k

ð2Þ

At isothermal–isobaric conditions (Section 2.2), k and D are timeindependent and Eq. 1 can be analytically integrated: −t

A = A0 10 D

ð3Þ

where A0 represents the initial activity. In the case of HT treatment at atmospheric pressure, the meaning of A0 is straightforward, it is the activity of the untreated enzyme. At HPHT conditions, on the other hand, A0 is defined as the activity of the sample taken after the initial

dynamic phase, at the start of isothermal–isobaric conditions (Section 2.2.2). In a second step, the impact of the process parameters pressure and temperature on the decimal reduction time was determined. The temperature dependence at constant pressure is expressed in terms of the zT value (°C), which is defined as the temperature increase necessary to obtain a 10-fold decrease of the D value. Tref −T

D = DrefT 10

ð4Þ

zT

where DrefT is the decimal reduction time at reference temperature Tref (K). Analogously, a log-linear relationship between the D value and pressure is also often found (Van Loey et al., 2002), whereby zP (MPa) is defined as the pressure increase necessary to obtain a 10-fold decrease of the D value. Pref −P zP

ð5Þ

D = DrefP 10

with DrefP the inactivation rate constant at reference pressure Pref (MPa). All kinetic parameters were estimated by non-linear regression analysis (SAS 9.1, USA). The suitability of the models selected (Eqs. 3, 4 and 5) and the quality of parameter estimation were visually assessed by inspecting parity plots (estimated values versus measured values of residual activity) and residue plots (residuals versus measured values) for the presence of any trends, not explained by the kinetic model. The adjusted R2 (R2adj) was used as a measure for the goodness of fit: 2 Radj

= 1−

  SSQregression ðm−1Þ 1− SSQ total

ðm−jÞ

ð6Þ

where m is the number of observations, j the number of model parameters and SSQ the sum of squares. 2.4.2. Validation of the kinetic model under dynamic conditions The validity of the kinetic inactivation model and its corresponding parameters, determined under isothermal–isobaric conditions, was verified under dynamic processing conditions. To this end, different dynamic pressure temperature profiles were applied with variable processing times, leading to a broad range of residual activities. The selected kinetic model (Eq. 4) with its corresponding parameters (Section 3.2.2), together with Eq. 2, were inserted in Eq. 1 and the

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3. Results and discussion 3.1. Assessment of indicator requirements 3.1.1. User-friendly with an accurate and reproducible read-out of an irreversible response A basic requirement for a pTTI is its user-friendliness. On condition that XTMB is available on the market, the indicator is easily and quickly prepared since this only implies preparation of an enzyme solution and enclosure in a recipient suitable for HPHT treatment. Furthermore, the indicator's response, i.e. its residual activity (%) after treatment, should be easily determined by an assay that gives accurate and reproducible results. To reliably quantify the process impact on the enzyme, the activity assay should give a clear and linear relationship between absorbance and enzyme concentration, so that a processing-induced decrease in activity is characterized by a proportional decrease in the response measure (absorbance). During optimization of the activity assay, it was found that addition of bovine serum albumin (BSA) to the reaction medium caused an increase in the activity of XTMB and a linearity up to a higher concentration of the enzyme (data not shown). BSA is frequently used to stabilize purified enzymes and to improve xylanase assays (Winterhalter & Liebl, 1995; Scheibe et al., 2007), nevertheless an explanation for these effects remains forthcoming. Scheibe et al. (2007) suggested that BSA probably acts unspecifically and by stabilization of the active conformation of the enzyme, leading to a reaction enhancement. After addition of BSA and upon further optimization, the activity assay ensured a good reproducibility (1.3% experimental error) and a detection limit of 1% residual activity of untreated sample. 3.1.2. Heat-stable at atmospheric pressure For application as an indicator in HPHT processes, XTMB must exhibit a considerable thermal stability at atmospheric pressure. In practice, a pTTI is inserted in a HP vessel already set at a relatively high temperature (60–90 °C), so a substantial activity loss before pressure build-up is undesirable. A screening of the thermal stability of XTMB (in 0.2 M sodium acetate buffer pH 5.0) was performed by a 5 min treatment at constant temperature. In accordance to the work of Ihsanawati et al. (2005), Jiang et al. (2001), Simpson et al. (1991), Sunna et al. (1997) and Winterhalter and Liebl (1995), the enzyme proved to be quite heat-stable: inactivation started only at 96 °C and 5 min at 106 °C caused a complete activity loss (data not shown). This means that barely any inactivation will occur in the hot HP vessel before pressure build-up. Furthermore, since this inactivation window merely concerns a temperature difference of 10 °C, XTMB is extremely sensitive to temperature alterations in this temperature range, which is a beneficial characteristic for indicators with a view to detect temperature inhomogeneities. 3.1.3. Temperature-sensitive response in the pressure–temperature window of HP sterilization In order to assess the temperature distribution in a HP vessel at HPHT conditions, an indicator should obviously display a temperature-sensitive response in this pressure–temperature window. To date, no open literature data are available on the pressure stability of XTMB, nor of any other xylanase. However, based on its thermophilic origin, a high pressure stability is to be expected (Eichler, 2001; Mombelli et al., 2002). The stability of XTMB was screened at 700 MPa in the temperature range of 104–114 °C for a treatment time of 5 min

(Fig. 2). XTMB showed to be extremely resistant to HPHT combinations; little or no inactivation occurred after 5 min at 700 MPa and 114 °C. In order to obtain a detectable change in response in the pressure– temperature range of HP sterilization, a destabilization of the enzyme was inevitable. It was found that addition of urea to the acetate buffer, used for preparation of the treatment solution, provided the intended result, without compromising the fulfillment of the first requirement for candidate indicators. In the presence of 2 M urea, XTMB still retained 59% of its initial activity after a treatment of 5 min at 700 MPa and 114 °C, but addition of 4 M urea resulted in an activity decrease up to 14% residual activity (Fig. 2). An additional observation was a rise in the initial activity with increasing concentrations of urea (data not shown). A possible explanation for this might be that urea causes a partial unfolding, which leads to a more open and flexible structure of the enzyme, possibly at the active site, resulting in an enhancement of the enzyme activity. Although the mechanism of action of urea in the unfolding of proteins is still unclear, Kumar et al. (2003) demonstrated that subtle structural changes in the secondary and tertiary structure of Chainia xylanase, upon addition of relatively low concentrations of urea, increased the enzyme activity. In this way, urea lowered the activation energy through which the xylanase achieved a higher catalytic rate. In case of a candidate indicator, this increase in initial activity is an interesting feature, since a relative activity decrease is taken into consideration as a response. As 4 M urea satisfactory lowered the stability of XTMB to the pressure–temperature range of HP sterilization, this condition was further examined. This enzyme system will be further referred to as XTMB-4M. In Fig. 3, the thermal stability of XTMB-4M at 500, 600 and 700 MPa is compared to that at atmospheric pressure. At atmospheric pressure, XTMB-4M still retained a sufficient heat stability that would allow application as an indicator in industrial HPHT processes. In the presence of 4 M urea, the inactivation range was shifted to a lower temperature range (85–102 °C for 5 min treatment), though not to the extent that any significant loss in activity would occur before pressure build-up. Application of pressure stabilized the enzyme against heat inactivation as the inactivation range shifted to higher temperatures. This is consistent with the statement of Hei and Clark (1994) and Mombelli et al. (2002) that thermophilic proteins are often stabilized by pressure. It is clear that in the presence of 4 M urea, there is a distinct and very temperature-sensitive response of XTMB-4M as a candidate indicator in the desired pressure–temperature window of HP sterilization. Furthermore, inactivation of XTMB-4M was found to be little or not pressure-sensitive in the explored window at elevated pressure. This was not regarded as an issue, since pressure is 100 90 80 70

A/Au (%)

differential equation was solved by numerical integration of the registered pressure temperature profiles, rendering the predicted residual activity. These estimated values were compared with the experimentally measured residual activities by examination of the parity and residue plot and calculation of R2.

191

60 50 40 30 20 10 0 104

109

114

Te (°C) Fig. 2. Screening of the thermal stability of XTMB at 700 MPa for a treatment time of 5 min (tiso) and different concentrations of urea: 0 ( ), 2 ( ) and 4 M (□).

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90

Table 1 Estimated decimal reduction times (± approx. standard error) for isothermal inactivation of XTMB-4M by the first-order model at atmospheric pressure.

A/Au (%)

80 70

T (°C)

D (min)

R2adj

60

85.0 88.0 89.0 90.0 91.0 92.0 93.0 94.0 96.0 98.0 100.0 102.0

86.8 ± 3.9 61.5 ± 2.8 53.8 ± 3.9 43.9 ± 1.7 42.3 ± 3.2 36.8 ± 0.9 28.2 ± 1.6 22.3 ± 0.9 15.1 ± 0.6 9.6 ± 0.4 5.8 ± 0.1 3.7 ± 0.2

0.997 0.998 0.995 0.999 0.995 0.999 0.997 0.998 0.998 0.997 0.999 0.995

50 40 30 20 10 0 60

70

80

90

100

110

120

T (°°C) Fig. 3. Screening of the thermal stability of XTMB-4M at 0.1 (♦), 500 ( 700 MPa (×) for a treatment time of 5 min (tiso).

), 600 (○) and

considered uniform and easily measurable in HP processes, and it is far more crucial for an indicator to display a temperature-sensitive response so that difficult to measure inhomogeneities in temperature can be detected. Because of the desired temperature-sensitivity of XTMB-4M's inactivation in the pressure–temperature window of HP sterilization, the condition of 4 M urea in 0.2 M sodium acetate buffer pH 5.0 was selected for preparation of the treatment solutions for further steps in the development of the indicator. 3.2. Kinetic calibration Since the requirements for a candidate indicator for HPHT processing were successfully met, in a next step, a detailed kinetic study was performed at HT as well as at HPHT conditions, in either case under isothermal–isobaric conditions. 3.2.1. Thermal inactivation at atmospheric pressure Based on the screening results of XMTB-4M's heat stability at atmospheric pressure (Fig. 3), the kinetics of irreversible inactivation were investigated in the temperature domain in which the largest activity decrease occurred (85–102 °C). In Fig. 4, the residual activity as a function of treatment time at constant elevated temperature is shown. These data were best fitted by a first-order model and no deviant trends were observed in parity and residue plots (results not

shown). Table 1 presents the results of non-linear regression for estimation of the decimal reduction times. The strong decrease in D values with increasing temperature confirms the high temperature sensitivity determined during screening (Section 3.1). The temperature dependence of the D values could be described by the TDT concept (Eq. 4). When the 10-based logarithm of the decimal reduction time is plotted against temperature (TDT plot), two zones of different temperature dependency are revealed (Fig. 5) as the straight line is bent at 91 °C. Apparently a change in inactivation kinetics occurs at this temperature. Nevertheless, the TDT model holds in each of the two temperature regions, yet with different DrefT and zT values (Table 2). It is clear that the D values exhibit a large difference in temperature sensitivity in the two regions. Where a zT value of 18.4 °C holds at temperatures lower than 91 °C, a value of only 10.6 °C was found at higher temperatures. No record could be found in literature of similar observations for xylanases nor other enzymes and no further explanation could be given. 3.2.2. Thermal inactivation at high pressure The kinetics of irreversible inactivation of XTMB-4 M by combined HPHT treatments were studied at 3 pressure (500, 600 and 700 MPa) and 4 temperature levels (99.4, 104.2, 109.0 and 113.8 °C). Similar to the inactivation at atmospheric pressure, the decrease in residual activity could again be best described by a first-order model. This is graphically depicted in Fig. 6. No deviant trends were observed in parity and residue plots (results not shown). The corresponding decimal reduction times are listed in Table 3. From these results, it is clear that the decimal reduction times are very sensitive to changes in temperature, but almost indifferent to 2.5

100 90

2.0

80

60

log(D)

A/Au (%)

70

50 40

1.5

1.0

30 20 0.5 10 0 0

20

40

60

80

100

120

140

time (min)

0.0 80

85

90

95

100

105

110

115

120

T (°C) Fig. 4. Time dependent changes in residual activity of XTMB-4M for isothermal inactivation at atmospheric pressure and 85 (♦), 88 ( ), 89 (×), 90 ( ), 91 (○), 92 ( ), 93 (+), 94 ( ), 96 (□), 98 ( ), 100 (Δ) and 102 °C (⁎). The full lines represent the firstorder model fit.

Fig. 5. TDT plot describing the temperature dependence of the decimal reduction time for isothermal–isobaric inactivation of XTMB-4M at 0.1 (♦), 500 ( ), 600 (+) and 700 MPa (○).

L. Vervoort et al. / Innovative Food Science and Emerging Technologies 12 (2011) 187–196 Table 2 Estimated kinetic parameters DrefT and zT (± approx. standard error) for the TDT model (Eq. 4) describing the temperature dependence of the decimal reduction time for isothermal inactivation of XTMB-4M at atmospheric pressure. A reference temperature of 90 °C was chosen for the lower temperature region and 100 °C for the upper region.

DrefT (min) zT (°C) R2adj

85–91 °C

91–102 °C

46.7 ± 0.3 18.4 ± 0.3 0.999

6.2 ± 0.1 10.6 ± 0.1 0.999

Table 3 Estimated decimal reduction times (± approx. standard error) for isothermal–isobaric inactivation of XTMB-4M by the first-order model at 500, 600 and 700 MPa. T (°C)

700 MPa

R2adj

D (min)

R2adj

D (min)

R2adj

48.9 ± 1.3 25.6 ± 1.0 14.0 ± 0.5 6.7 ± 0.3

0.998 0.997 0.995 0.992

48.7 ± 1.9 26.1 ± 1.3 13.1 ± 0.7 6.2 ± 0.5

0.998 0.997 0.995 0.992

43.6 ± 2.6 24.0 ± 2.1 13.1 ± 1.2 6.2 ± 0.4

0.995 0.990 0.990 0.992

100

600 MPa

90

80

80

70

70

60

60

A/Au (%)

A/Au (%)

600 MPa

D (min)

Furthermore, it can be stated that in the investigated domain of elevated pressures, there is little or no mutual difference between the DrefT and zT values corresponding to the different pressure levels. In terms of the TDT model, this small pressure dependence implies an extremely large zP value: estimates between 4000 and 8000 MPa were obtained (Eq. 5), depending on the temperature level. However, the reliability of this parameter estimation can be questioned, because it involves a strong extrapolation since the experimental domain only covers a much smaller pressure range of 200 MPa. Given this negligible pressure dependence, only the temperature dependence of the D values was further taken into account and the kinetic parameters were re-estimated combining the data of the three pressure levels. Description of the residual activity decrease by the first-order model displayed no deviant trends in parity and residue plots (results not shown). The corresponding decimal reduction times are listed in Table 5, while Table 6 presents an overview of the DrefT and zT values for the TDT model at atmospheric

500 MPa

90

500 MPa

99.4 104.2 109.0 113.8

changes in pressure. At a constant pressure, the D value decreases with increasing temperature, indicating a faster inactivation. This temperature dependence could be described by the TDT model (Eq. 4). The DrefT and zT values derived from this equation are summarized in Table 4. As was expected from the screening results on the stability of XTMB-4M at HPHT conditions (Section 3.1.3), the D value at reference temperature is significantly higher at elevated pressure, in comparison to the value at ambient pressure in the upper temperature region (compare with Table 2, right column). This results in a shift of the TDT curve to higher temperatures (Fig. 5). When comparing the zT values, it is clear that the temperature dependence is less pronounced at elevated pressure than in the temperature range of 91–102 °C at ambient pressure, but only slightly different from the one in the range of 85–91 °C. Indeed, the TDT curves at elevated pressure display a slope comparable to the part of the curve at 85–91 °C and atmospheric pressure.

100

193

50 40

50 40

30

30

20

20

10

10

0

0 0

10

20

30

40

50

0

60

10

20

tiso (min)

30

40

50

60

tiso (min)

100

700 MPa

90 80

A/Au (%)

70 60 50 40 30 20 10 0 0

10

20

30

40

50

60

tiso (min) Fig. 6. Time dependent changes in residual activity of XTMB-4M for isothermal–isobaric inactivation at elevated pressure and 99.4 (♦), 104.2 (+), 109.0 (▲) and 113.8 °C (○). The full lines represent the first-order model fit.

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Table 4 Estimated kinetic parameters DrefT and zT (± approx. standard error) for the TDT model (Eq. 4) describing the temperature dependence of the decimal reduction time for isothermal–isobaric inactivation of XTMB-4M at 500, 600 and 700 MPa and at a reference temperature of 100 °C.

DrefT (min) zT (°C) R2adj

500 MPa

600 MPa

700 MPa

45.1 ± 0.1 17.2 ± 0.1 0.999

45.0 ± 0.1 16.8 ± 0.1 0.999

40.5 ± 0.1 17.9 ± 0.1 0.999

pressure (left) and at elevated pressure (right). The validity of this model and its parameters was subsequently verified by evaluation of the integrating properties under variable pressure temperature conditions (Section 3.3). A unique characteristic of XTMB-4M is that the temperature sensitivity at HPHT matches that of recently reported spore inactivations of Clostridium sporogenes in various foods (Zhu et al., 2008; Shao & Ramaswamy, 2008; Ramaswamy et al., 2010). Although a target microorganism for HP sterilization has not been determined to date, the use of C. sporogenes as surrogate microorganism for assessment of the HP sterilization impact can be justified, because of the spores' high resistance to HPHT conditions (Institute of Food Technologists (IFT), 2000; Zhu et al., 2008). Furthermore, it has a strong genetic resemblance to proteolytic Clostridium botulinum, the target organism for heat-sterilized foods (Sebaihia et al., 2007). Unfortunately, literature data on the latter's spore inactivation kinetics at HPHT are scarce. Some data have been published for an extremely stable C. botulinum strain TMW 2.357, suggesting a higher resistance to HPHT than C. sporogenes and kinetics which deviate from the first-order model, found for inactivation of C. sporogenes and XTMB-4M (Margosch et al., 2006). However, it must be noted that this inactivation study was done in Tris (tris(hydroxymethyl)aminomethane)–HCl buffer, a well-known temperature-sensitive buffer (Dawson et al., 1969) and that the strain also exhibits a higher thermal stability at atmospheric pressure than the strains used to establish the 12-D concept for thermal sterilization. The comparable zT value of XTMB-4 M to that of C. sporogenes suggests the possibility that, next to utilization as an indicator for temperature gradients, the application of XTMB-4M as an indicator for process impact evaluation of HP sterilization processes could also be within reach (Van der Plancken et al., 2008). However, it should be noted that it is only the temperature sensitivity that matches, as zP values between 450 and 1250 MPa at 80–100 °C were estimated for C. sporogenes (Zhu et al., 2008; Shao & Ramaswamy, 2008; Ramaswamy et al., 2010). But, as mentioned before, pressure can easily be registered during the process and is considered uniform (Section 1). Before this application of XTMB-4M can be further investigated, extensive research is necessary on the target microorganism for HP sterilization, the pressure and temperature sensitivity of its spore inactivation and the pressure and temperature sensitivities of different surrogates, as small differences between the surrogates and target can be very important.

Table 5 Estimated decimal reduction times (± approx. standard error) for isothermal–isobaric inactivation of XTMB-4M by the first-order model at elevated pressure (500–700 MPa), leaving the pressure dependence out of consideration. T (°C)

99.4 104.2 109.0 113.8

500–700 MPa D (min)

R2adj

47.3 ± 1.2 25.1 ± 0.9 13.4 ± 0.4 6.3 ± 0.2

0.996 0.994 0.990 0.987

Table 6 Summary of the estimated kinetic parameters DrefT and zT (± approx. standard error) for the TDT model (Eq. 4) describing the temperature dependence of the decimal reduction time for isothermal–isobaric inactivation of XTMB-4M, leaving the pressure dependence at elevated pressure out of consideration. A reference temperature of 90 °C was chosen for the lower temperature region at atmospheric pressure and one of 100 °C for the upper region at atmospheric pressure and for HPHT conditions. 0.1 MPa

DrefT (min) zT (°C) R2adj

500–700 MPa

85–91 °C

91–102 °C

99.4–113.8 °C

46.7 ± 0.3 18.4 ± 0.3 0.999

6.2 ± 0.1 10.6 ± 0.1 0.999

43.7 ± 0.1 17.2 ± 0.1 0.999

3.3. Validation of the kinetic model under dynamic conditions In industrial HP applications, isothermal–isobaric conditions are usually not encountered (Section 1). Therefore, it is indispensable to evaluate the validity of the kinetic inactivation model (Eq. 4) and its corresponding parameters (Table 6), determined at isothermal– isobaric conditions, under dynamic processing conditions. To this end, different combinations of dynamic pressure and temperature profiles were applied with variable processing times, leading to a broad range of process intensities. The resulting residual activities were experimentally measured and compared to the values estimated by numerical integration of the recorded temperature profiles using Eq. 7 (obtained by insertion of Eqs. 2 and 4 into Eq. 1): dA =− dt

ln ð10Þ Tref −T

DrefT 10

A

ð7Þ

zT

Before pressure build-up, upon insertion in the pressure vessel, the parameters estimated at atmospheric pressure were applied (Table 6, left). At the start of pressure increase, the parameters estimated at 500–700 MPa were used (Table 6, right), since the thermal inactivation of XTMB-4M is faster at atmospheric pressure than at elevated pressure and process impact should never be overestimated. A high correlation between the measured and predicted residual activities was observed (Fig. 7) (R2 = 0.980) and the residue plot revealed no deviant trends (results not shown), indicating the applicability of the kinetic model under dynamic processing conditions, relevant for industrial HPHT applications. This demonstrates the power of the determined model to predict the read-out of the indicator after a particular dynamic HPHT treatment. 4. Conclusion The time- and position-dependent temperature gradients throughout the pressure vessel and the product load are an important issue that should be taken into account in refining process and equipment designs within the scope of HP sterilization of foods. In this work, an enzymatic indicator was developed as a tool to map out the temperature non-uniformity. Since enzymes from thermophilic microorganisms show good potential, xylanase B from T. maritima was selected as a possible candidate indicator. It was the first time that the pressure sensitivity of this enzyme was studied. XTMB-4M met the three requirements of sensors for the detection of temperature inhomogeneities in the pressure–temperature window of HP sterilization: (1) it is user-friendly and its read-out showed a good accuracy and reproducibility, (2) it proved to be quite heatstable at atmospheric pressure, so that barely any inactivation will occur during insertion in a hot HP vessel before pressure build-up, and (3) XTMB-4M was very sensitive for temperature differences in the processing window of HP sterilization.

L. Vervoort et al. / Innovative Food Science and Emerging Technologies 12 (2011) 187–196

100 90

A/Au estimated (%)

80 70 60 50 40 30 20 10 0

0

10

20

30

40

50

60

70

80

90 100

A/Au measured (%) Fig. 7. Validation of the kinetic model under dynamic conditions: correlation between the experimentally measured residual activity of XTMB-4M after a dynamic HPHT treatment and the residual activity estimated by numerical integration of the dynamic pressure– temperature profiles and the determined TDT model (Eq. 7) and its corresponding parameters (Table 6) (R2 = 0.980).

The thermal inactivation at isothermal–isobaric conditions could be described by a first-order model, at atmospheric pressure as well as at elevated pressure. At HPHT conditions, the pressure dependence of the D values was negligible, while the temperature dependence was distinct. This pressure independence suggests that a more accurate term for XTMB is ‘a TTI for HPHT applications’, rather than a pTTI. The TDT model entailed a zT value of 17.16 ± 0.04 °C, which matches the temperature sensitivity of C. sporogenes spores' inactivation. This could suggest the possible additional application of XTMB-4M as an indicator for process impact evaluation of HP sterilization processes. The inactivation model, with its corresponding parameters, was successfully validated under dynamic HPHT conditions, demonstrating its ability to predict the read-out of the indicator after a dynamic HPHT treatment. Future research involves the application of XTMB-4M in temperature uniformity studies in industrial HPHT equipment. In this way, the point of lowest heat impact in the vessel can be located. Acknowledgements This work was financially supported by the Research Fund of Katholieke Universiteit Leuven, the Fund for Scientific Research Flanders (FWO) and the Commission of the European Communities, Framework 6, Priority 5 “Food Quality and Safety”, Integrated Project NovelQ FP6-CT-2006-015710. References Barbosa-Cánovas, G. V., & Juliano, P. (2008). Food sterilization by combining high pressure and thermal energy. In G. F. Gutiérrez-Lopez, G. V. Barbosa-Cánovas, J. Welti-Chanes, & E. Parada-Arias (Eds.), Food Engineering: Integrated Approaches (pp. 9−46). New York: Springer. Bauer, B. A., & Knorr, D. (2005). The impact of pressure, temperature and treatment time on starches: Pressure-induced starch gelatinisation as pressure time temperature indicator for high hydrostatic pressure processing. Journal of Food Engineering, 68, 329−334. Black, E. P., Setlow, P., Hocking, A. D., Stewart, C. M., Kelly, A. L., & Hoover, D. G. (2007). Response of spores to high-pressure processing. Comprehensive Reviews in Food Science and Food Safety, 6, 103−119. Bricher, J. L. (2009). NCFST receives regulatory acceptance of novel food sterilization process. [online]. http://www.iit.edu/ncfst/news_and_events/media_room/pdfs/ PressReleasePATSFDAAccept.pdf Last accessed on 06/07/2010. Chen, C. C., Adolphson, R., Dean, J. F. D., Eriksson, K. E. L., Adams, M. W. W., & Westpheling, J. (1997). Release of lignin from kraft pulp by a hyperthermophilic xylanase from Thermatoga maritima. Enzyme and Microbial Technology, 20, 39−45. Claeys, W. L., Indrawati, Van Loey, A. M., & Hendrickx, M. E. (2003). Review: Are intrinsic TTIs for thermally processed milk applicable for high-pressure processing assessment? Innovative Food Science & Emerging Technologies, 4, 1−14.

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