Temperature uniformity mapping in a high pressure high temperature reactor using a temperature sensitive indicator

Journal of Food Engineering 105 (2011) 36–47

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Temperature uniformity mapping in a high pressure high temperature reactor using a temperature sensitive indicator Tara Grauwet a, Iesel Van der Plancken a, Liesbeth Vervoort a, Ariette Matser b, Marc 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 Wageningen UR Food and Biobased Research, P.O. Box 17, 6700 AA Wageningen, The Netherlands

a r t i c l e

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Article history: Received 5 May 2010 Received in revised form 24 September 2010 Accepted 3 January 2011 Available online 14 January 2011 Keywords: Pressure–temperature–time indicator (pTTI) High pressure high temperature (HPHT) processing Kinetics Temperature uniformity Ovomucoid

a b s t r a c t Recently, the first prototype ovomucoid-based pressure–temperature–time indicator (pTTI) for high pressure high temperature (HPHT) processing was described. However, for temperature uniformity mapping of high pressure (HP) vessels under HPHT sterilization conditions, this prototype needs to be optimized. To this end, this work aimed at the development of an ovomucoid-based indicator with combined pressure temperature dependent inactivation kinetics and a sufficient pressure temperature stability relevant for commercial HPHT sterilization. After varying buffer type and the pH at ambient pressure and temperature (pHi), an indicator based on 1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.2 was selected. The inactivation behavior of this indicator system is characterized by pressure temperature dependent (combined Arrhenius–Eyring) first-order kinetics in the processing domain relevant for HPHT sterilization. This indicator showed good integrating properties under isobaric–isothermal and dynamic pressure temperature conditions. In a temperature uniformity study of a vertically oriented, pilot-scale HPHT vessel, pTTI readouts at different coordinates illustrated low and high temperature zones. As the inactivation of spores under HPHT is clearly positively temperature dependent, the food safety objective has to be verified in the former sampling zone. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction 1.1. High temperature short time treatment of conductively heating products Enhancing microbial safety and extending the shelf-life of high moisture content food products is generally performed by relatively slow thermal processes (de Heij et al., 2005). The high temperatures needed, whether or not in combination with long residence times, result in a decrease in food quality (e.g. texture, nutritional value). The high temperature short time (HTST) principle has been introduced as a basis for the optimization of thermal sterilization. Today, liquid foods, whose temperature can be increased and decreased rapidly by exploiting convection phenomena, are successfully flash heated and cooled in industry resulting in high residual quality products after processing (e.g. UHT milk) (Holdsworth, 2009).

⇑ 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). 0260-8774/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.01.001

Recently, high pressure high temperature (HPHT) treatment (500–800 MPa; 80–120 °C; 1–10 min) of food products has been put forward as a worthy alternative for the optimization of sterilizing conductively heating (i.e. solid) food products (de Heij et al., 2003; Heinz and Knorr, 2005; Barbosa-Canovas and Juliano, 2008). In HPHT processing, compression and decompression of compressible materials causes respectively rapid heating and cooling of food products, since a temperature change is linked to every pressure change (Barbosa-Canovas and Rodriguez, 2005). As pressures can be generated fast, this phenomenon can create reduced process times (see HTST principle) (de Heij et al., 2005; Heinz and Knorr, 2005; Juliano et al., 2009a). The improved quality of conductively heating products after a HPHT treatment in comparison to their equivalently conventionally treated counterparts has been discussed (Matser et al., 2004; de Heij et al., 2005; Juliano et al., 2007; De Roeck et al., 2008, 2009; Leadley et al., 2008). In this context, a HPHT process is sometimes termed a ‘pressure-assisted thermal process’ (PATP) (Barbosa-Canovas and Juliano, 2008). Starting from room temperature, using only compression heating, the food product temperature cannot be raised to the point where inactivation of spores under high pressure (HP) is feasible. Therefore, a preheating step to a well-defined initial temperature

T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

(Ti) (e.g. Ti = 90 °C) needs to precede the actual HPHT treatment. When pressurization starts from this Ti, due to compression heating, the temperature of the product reaches the process temperature (Tp) (e.g. Tp = 121 °C for water-like components assuming adiabatic conditions during pressure build-up to 600 MPa). In practice, a HPHT process is a three-step process: (i) preheating at atmospheric pressure; (ii) actual HPHT treatment; (iii) further cooling at atmospheric pressure. 1.2. Hurdles in high pressure high temperature processing implementation In contrast to UHT-applications, to date, no commercial application of HPHT processing is available. In the literature, different hurdles have been reported: (i) insufficient insight of the temperature distribution in a HPHT reactor (Knoerzer et al., 2007; Juliano et al., 2009a) and poor understanding of the process uniformity of a HPHT treatment (Denys et al., 2000; Juliano et al., 2009b); (ii) HPHT food products are subjected to the ‘novel food regulation’ (EC 258/97) (Howlett et al., 2003); (iii) no industrial-scale HPHT unit has been built that incorporates all individual HPHT processing steps. In the following section, hurdle (i) will be focused on. 1.3. Insight in the temperature distribution in a HPHT reactor and its effect on the process impact uniformity Pressure, temperature and time are the critical process variables in HPHT processing (Barbosa-Canovas and Juliano, 2008; Ramaswamy et al., 2009; De Roeck et al., 2010). It has been generally acknowledged that high pressure used in HPHT processing can be assumed uniform. In addition, time is fixed as a HPHT process operates under batch conditions. However, securing temperature uniformity in HPHT reactors is not straightforward (Delgado and Hartmann, 2003). Differences in compression heat of pressurized materials in HP reactors (e.g. pressure medium, food components) and the corresponding heat transfer underlie the existence of temperature gradients in HP vessels (Delgado et al., 2008). Since the inactivation kinetics of spores, in view of HPHT sterilization, are clearly positively temperature dependent, it is very likely that temperature non-uniformity under HPHT conditions results in process impact non-uniformity (Margosch et al., 2006; Zhu et al., 2008; Shao et al., 2008). Direct monitoring of the temperature profile at different coordinates in a HPHT reactor seems to be the most straightforward method to gain insight in the temperature distribution in a HPHT reactor. However, this method is technically too complex at pilot or industrial scale. There is a need for another method easily detecting temperature differences under HPHT conditions. Once different temperature zones could be indicated, it would be necessary to increase the understanding on the effect of these different temperature zones on the process impact distribution. This requires either kinetic information of the target attributes under HPHT conditions or direct process impact evaluation on the target attribute (i.e. in situ method). Kinetic data of target attributes under HPHT conditions are scarce. As a first step in more removing hurdle (i), this work aimed at the development of a method for easy detection of low and high temperature zones in a HPHT vessel. 1.4. Protein-based pressure–temperature–time indicator (pTTI) for mapping temperature uniformity in a high pressure high temperature reactor In general, a pTTI can be defined as a small, wireless pressure temperature sensitive device characterized by an easily quantifiable, irreversible response to the HP treatment (Van Loey et al., 2002; Van der Plancken et al., 2008). The potential of two a-amy-

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lase-based pTTIs to detect low and high temperature zones in an industrial-scale vessel under HP pasteurization (HP-P) conditions (400–600 MPa; 10–40 °C; 1–15 min) was previously demonstrated (Grauwet et al., 2009, 2010a,c). However, the stability of these aamylase-based indicators does not allow use under HPHT conditions: the HP-P indicator read-out would be below the detection limit after HPHT treatment. Recently, the first prototype protein-based pTTI with potential use under HPHT conditions has been described (Grauwet et al., 2010b). A protein system consisting of 1 g/L ovomucoid, a commercially available inhibitor of trypsin, dissolved in a specific solvent environment (0.1 M sodiumphosphate buffer pH 8.0) (OMPhB8.0) was characterized by a temperature sensitive inactivation under increased pressure (p > 400 MPa) in the HPHT window (i.e. range of pressure–temperature–time conditions necessary for reaching HPHT conditions). The temperature sensitive inactivation under increased pressure suggested the potential of an ovomucoidbased system as a tool to map temperature uniformity under HPHT conditions. However, the prototype developed had some drawbacks: (i) pressure changes (400–700 MPa) did not affect the inactivation rate; (ii) the inactivation window of the system was restricted to mild HPHT conditions (400–700 MPa; Tp 95–110 °C; 0–20 min), in which spores inactivation occurs but is reduced; (iii) the thermal stability of the ovomucoid system at atmospheric pressure was rather limited. In this work, the first prototype sensor will be optimized using solvent engineering (purposely changing the solvent characteristics in order to reach the desired indicator characteristic) to accomplish the following objectives: (i) obtaining a pressure sensitive inactivation of ovomucoid, without restriction of the temperature sensitivity; (ii) shifting the ovomucoid inactivation window to the HPHT processing window relevant for commercial sterilization; (iii) improving the heat stability of the candidate indicator at atmospheric pressure. The potential of such a pressure temperature sensitive protein-based indicator to map temperature uniformity was experimentally verified. 2. Materials and methods For all data reported in this work, the same experimental approach was used. First, an ovomucoid-based indicator system was prepared by dissolving ovomucoid in a specific solvent conditions (Section 2.1). Next, this system was treated under particular pressure–temperature–time conditions (Sections 2.2 and 2.3). In a third step, its irreversible read-out upon treatment was quantified (Section 2.4). Finally, data obtained were analyzed (Section 2.5). In the following, materials and methods will be described step-bystep. 2.1. Ovomucoid-based indicator system Ovomucoid (EC 2329069) is a trypsin inhibitor present in chicken egg white. Type III-O (no ovo-inhibitor) was purchased in dried state from Sigma (lot number 117K7011, Germany). To avoid batch-to-batch differences, for all experiments performed, the same storage solution of ovomucoid was used as described by Grauwet et al. (2010b). From this storage solution (100 g/L in 0.1 M Tris (2-amino-2-hydroxymethyl-propane-1,3-diol)-HCl buffer pH 8.6), just before treatment, the ovomucoid system was prepared by dilution in a given buffer condition to a concentration of 1 g/L. The effect of initial pH (i.e. pH measured at atmospheric pressure and room temperature; pHi) of a pressure stable MES (2-(Nmorpholino)ethanesulfonic acid)-NaOH buffer (pH 5.0–7.0) on the inhibitor capacity of ovomucoid was investigated in the context of solvent engineering.

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2.2. High pressure high temperature treatment The ovomucoid systems (1 g/L – various solvent environments) were pressure treated in flexible microtubes made from polyethylene terephthalate (250 lL, 0.5  3 cm, Carl Roth, Germany), which transmit pressure and temperature easily. After the treatment, samples were immediately transferred to an ice bath to prevent further denaturation. Results and discussions can be devided in two parts: one part describing the development of the indicator (Sections 3.1–3.4) and one parts describing the application of the indicator (Section 3.5). For the development of the indicator, the lab-scale HPHT unit was used (Section 2.2.1). The potential of the indicator developed to detect temperature differences was investigated using a larger-scale equipment (Section 2.2.2). Today, HPHT processing only reached pilot-scale. 2.2.1. Lab-scale high pressure high temperature equipment A laboratory scale, multi-vessel, HP equipment (custom-made, Resato, The Netherlands) was used consisting of six individual, vertically oriented vessels (Vvessel = 0.043 L; Øvessel = 2 cm), surrounded by an isolated heating coil connected to a heating/cooling unit. This equipment allows computer-controlled pressure build-up up to 800 MPa and temperature control up to 120 °C. In this lab-scale equipment, direct monitoring of the pressure temperature profile of the sample is possible. The pressure medium was 100% propylene glycol (PG fluid, Resato, The Netherlands). Pressure is increased by pressure medium addition at the vessel bottom (indirect compression; San Martin et al., 2002). Because this system is computer controlled, treatments were highly repeatable. This equipment was used in two stages of the development: the screening of the effect of solvent conditions and the calibration of the kinetics of the selected indicator system under isobaric–isothermal conditions (Sections 3.1 and 3.2) and the validation of the kinetic model obtained in Section 3.2 under dynamic pressure temperature conditions (Section 3.3). 2.2.1.1. Experiments under isobaric–isothermal conditions. A protocol was established to obtain isobaric–isothermal pressure temperature conditions in time and space in the HPHT domain. In this protocol, different steps of the HPHT process (e.g. preheating, actual HPHT treatment, cooling phase) were included. Based on the combination of two methods reported in the literature for blocking temperature gradients in a HP vessel, isobaric–isothermal conditions could be reached: (i) the use of a cylindrical, poly-oxy-methylene polymer (POM) sample holder with insulator capacities (Knoerzer et al., 2007; Juliano et al., 2009b) and (ii) the control of the temperature of the pressure medium in the sample holder on the one hand and the temperature of the vessel wall and the temperature of the pressure medium in the vessel on the other hand (Rauh et al., 2009). The protocol applied was previously described in detail and graphically represented by Grauwet et al. (2010b). The volume of the water in the sample holder is approximates 30 mL. Depending on the pressure level applied, the volume of the PG medium in the vessel, when the vessel is filled with the sample holder is approximately 12 mL. Pressure was built up at a high rate (from 0.1 to 150 MPa in 2 s and furthermore to the set pressure at 10 MPa/s) to the holding pressure. After attaining the desired pressure, the individual vessels were isolated and an equilibration period of 1.5 min was taken into account to ensure isobaric–isothermal conditions (Grauwet et al., 2010b). After this dynamic phase, the pressure of a first vessel was released. The corresponding sample was considered as the reference sample (tiso = 0 min). The importance of performing kinetic experiments under isobaric–isothermal pressure temperature conditions has been reported in the literature (Shao et al., 2008; Ramaswamy et al., 2009): only data obtained under isobaric–isothermal pres-

sure temperature conditions can be interpreted independently from and extrapolated to another equipment design. In this work, processing conditions defined by isobaric pressures of 500– 700 MPa in combination with isothermal temperatures (i.e. process temperatures) of 107–119.5 °C were studied. 2.2.1.2. Validation experiments under dynamic pressure temperature conditions. During the dynamic treatments, ovomucoid systems were treated directly in the PG fluid, which was equilibrated at a given vessel temperature. To this end, ovomucoid systems were attached to a thermocouple (type J; 36.8 mm) at the center of the vessel closure. Next to temperature, pressure was logged. The protocol used was previously described in detail and graphically represented by Grauwet et al. (2010b). Pressure was built up immediately (2 s) to 150 MPa and subsequently at different pressure build-up rates (2–12.5 MPa/s) to the preset pressure. For these dynamic treatments (x = 96), a holding pressure range of 500– 700 MPa, an initial temperature range (Ti) of 80–95 °C (Ti is temperature of vessel wall and pressure medium at the start of the pressure treatment) and treatment times (tdyn) 0–20 min were used. 2.2.2. Pilot-scale high pressure high temperature equipment The potential of the candidate indicator to map temperature uniformity based on the evaluation of its indicator readings after treatment was studied at pilot-scale. The HPHT equipment used (developed by Resato, Solico, Unilever and Wageningen UR Food and Biobased Research, The Netherlands), consists of a preheating unit (product immersion), a single, vertically oriented HPHT vessel of large volume (Vvessel = 2.5 L; Øvessel = 10 cm) and a cooling unit (product immersion). During the actual HPHT treatment, pressure is increased up to 800 MPa due to volume reduction using a plunger at the vessel top (direct compression; San Martin et al., 2002). Typically, pressure can be built up to 700 MPa in 24 s. The temperature of the vessel is controlled by an electric heating jacket attached to the outer vessel wall and a bottom heater to heat up the vessel wall to a maximal temperature of 90 °C. The pressure medium used is tap water. Heat flow, and thus build-up of temperature gradients, from pressurized and heated content of the vessel under pressure (Tcontent > Ti due to compression heating) towards the vessel wall (Twall = Ti due to negligible compressibility) is retarded by the use of a POM-liner at the inner vessel wall and an isolating POM-sample container. This container is dimensioned to optimally fill the vessel (douter = 9.5 cm; louter = 36 cm) and has a movable cap to transmit the pressure. The system is computer controlled and process conditions (p, T, t) are automatically logged. However, only temperature at the outer wall can be logged, which is assumed to be equal to the Ti. The process temperature (i.e. temperatures reached under HP starting from Ti) cannot be controlled. 2.2.2.1. pTTI positioning. In the pilot-scale HPHT unit used, samples are treated in a cylindrically shaped sample holder (douter = 8.4 cm; louter = 29.4 cm). The sample holder is provided with plenty of holes so the pressure medium can easily spread over the whole volume of the vessel. Using this sample holder and ty-raps, indicator tubes could be easily fixed at different coordinates of the HP vessel. By positioning six pTTIs at different distances from the vessel bottom (3.5 cm (bottom (b)); 16 cm (middle (m)); 29 cm (top (t)) and different distances from the vessel wall (0.8 cm (wall (w)); 5 cm (center (c)), the effect of axial and/or radial coordinates on the indicators’ read-out was investigated. In Fig. 1, a schematic set-up is shown. 2.2.2.2. High pressure high temperature process. First, the isolating sample container (no content) was 15 min preheated in the preheating unit equilibrated at the initial temperature of the process condition under consideration to ensure a homogeneous temperature. In parallel, after positioning the pTTIs in the sample holder

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(see above), the pTTIs were preheated during exactly 4 min in the preheating unit. After 4 min, the sample holder with pTTIs was put in the sample container, filled with water from the preheating unit. Subsequently, the filled sample container was transferred manually to the emptied HPHT vessel which was already equilibrated at initial temperature. In a first phase of the pressure cycle, the HPHT vessel was filled with water from the prefill unit (equilibrated at the initial temperature selected), in a second phase, pressure was built up at the rate of 30 MPa/s using the plunger. After a selected holding time, pressure was released and the sample holder and content were removed manually and were transferred to the cooling bath (20 °C). In this work, processing conditions defined by an initial temperature of 85 °C, a holding pressure of 600 MPa and holding times of 2 and 5 min were selected. Each processing condition was tested in triplicate. During the treatments performed, bottom heating was turned off to provoke temperature gradients and to evaluate the potential of the pTTI to detect temperature non-uniformities. 2.3. Thermal treatment The thermal sensitivity at ambient pressure of the ovomucoid systems selected based on pressure treatments was investigated in the temperature range of 90–130 °C. Isothermal treatments were performed in a temperature controlled oil bath (Thermo Scientific, The Netherlands) using glass capillaries (Hirschmann Labogeräte, Germany) to ensure instantaneous heating and cooling of the ovomucoid solution inside. After treatment, samples were immediately transferred to an ice bath to prevent further denaturation. 2.4. Determination of the trypsin-inhibitor activity The trypsin-inhibitor activity of ovomucoid was quantified indirectly by trypsin-activity (TA in BA U/mg) measurement of an ovomucoid-trypsin mixture: for example, if the inhibitor capacity decreased due to ovomucoid denaturation, a higher trypsin activity of the ovomucoid-trypsin mixture was observed. In

Fig. 1. Schematic overview of pTTI positioning throughout the HPHT vessel (dashed line: POM container; bold line: sample holder). Different distances from the vessel bottom (3.5 cm (bottom (b)); 16 cm (middle (m)); 29 cm (top (t)) and different distances from the vessel wall (0.8 cm (wall (w)); 5 cm (center (c)) were studied.

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this work, a decrease in inhibitor capacity of ovomucoid will be termed ‘ovomuocoid inactivation’. The trypsin-inhibitor activity of each ovomucoid system was determined using an optimized spectrophotomethric assay as described by Grauwet et al. (2010b). This assay is based on the action of (partially inhibited) trypsin on a Na-Benzoyl–Arginine–Ethyl–Ester substrate (BAEE) (trypsin quality control test of Sigma, Germany). Trypsin cleaves the ester bond releasing Na-benzyol-L-arginine units (BA in U/ mg), which absorbs light at 253 nm (trypsin quality control test of Sigma, Germany). This results in an increase in absorbance at 253 nm (DA253 nm), which was recorded during 3 min at 25 °C. The slope DA253 nm/min of the linear section of this curve was a measure for the trypsin activity; a lower slope value corresponds to a higher inhibitor capacity (Van der Plancken et al., 2004). Based on the ratio of the trypsin activity of trypsin-ovomucoid mixtures and the trypsin activity of trypsin, the trypsin-inhibitor activity (TIA, %) and the residual trypsin-inhibitor activity (resTIA, %) could be calculated (Van der Plancken et al., 2004; Grauwet et al., 2010b). The detection limit for the residual trypsin-inhibitor activity response was 6%. All samples were measured twice. Averaged resTIA-values were interpreted. The absolute experimental error was 2%. 2.5. Data analysis 2.5.1. Inactivation data obtained under isobaric–isothermal conditions Kinetic parameters of the inactivation of ovomucoid under isobaric–isothermal HPHT conditions were estimated using a 2-step regression approach. In a first step, the time-dependent inactivation was modeled. The ability of a first-order model to describe the time-dependent inactivation of ovomucoid under pressure and temperature has been reported previously (Grauwet et al., 2010b)

resTIA ¼ resTIA0 expðktiso Þ

ð1Þ

where resTIA and resTIA0 represent the residual trypsin-inhibitor activity at treatment time tiso (min) and at treatment time tiso = 0 min, respectively and k the reaction rate constant (min1) at isobaric–isothermal conditions. Inherent to the HPHT protocol used, before reaching isobaric–isothermal conditions, indicator systems experienced a certain prehistory (see Section 2.2.1). Consequently, the resTIA0 does not necessarily equal to 100%. In a second step, the sensitivity of the reaction rate constant, k, to temperature or pressure was assessed. The temperature dependency of k at constant pressure was evaluated using the Arrhenius equation (Arrhenius, 1889), while its pressure dependency at constant temperature was evaluated by the Eyring equation (Eyring, 1946). In food processing, it is common to characterize first-order reactions in terms of the decimal reduction time (DT and Dp-value in min at constant pressure and temperature, respectively) and zT(°C) and zp- (MPa) values (i.e. temperature/pressure change required at constant pressure/temperature to achieve a ten-fold change in the D-value, respectively) by the use of the Thermal Death Time model (Bigelow, 1921). Likewise, parameters were obtained through non-linear regression analysis (SAS, version 9.2, USA). In addition, different models predicting kT,p as a function of both pressure and temperature were fitted to the data to investigate the combined pressure and temperature dependency of the inactivation rate constant (for example as used by Van Eylen et al. (2007), Buckow et al. (2009) and Katsaros et al. (2009)). Using a step-wise multivariate regression analysis, significance analysis on the different terms was performed. By combination of both the Arrhenius and the Eyring equation, a combined Arrhenius–Eyring model could be built (Eq. (2)):

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kT;p

T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

     Ea 1 1 V a exp ¼ kref T;p exp  ðp  pref Þ R T T ref RT

ð2Þ

For all different modeling steps under isobaric–isothermal conditions, model discrimination and evaluating were performed based on R2adjusted (Eq. (3)), root mean squared error (RMSE; Eq. (4)), R2normal probability and visual inspection of the parity plot, the scatter plot of the residuals versus the predicted values, the lag plot of the residuals versus residuals and the normal probability plot of the residuals



1

SSQ regression SSQ total

 ðx  1Þ

R2adj ¼ 1  ðx  p0  1Þ sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi MSQ residuals RMSE ¼ x  p0

ð3Þ

3.1. Screening the effect of solvent conditions on the pressure temperature sensitivity of the protein system In general, in the second step of indicator development (Grauwet et al., 2009), the effect of solvent conditions on the pressure temperature sensitivity of the protein system are screened in order to select the solvent conditions in which the protein shows the most interesting pressure temperature stability as well as sensitivity. As indicated above, in this work, solvent engineering, in particular the effect of buffer type and pHi, was studied to accomplish the three objectives. The first objective was to select a solvent condition in which ovomucoid shows a combined pressure and temperature sensitive inactivation.

ð4Þ

3. Results and discussion

3.1.1. Effect of buffer type In general, a distinction can be made between temperature–stable (e.g. phosphate, citrate) and pressure-stable (e.g. MES-NaOH, TrisHCl) buffers, whose pKa is stable as a function of temperature and pressure, respectively (Bates, 1962; Kitamura and Itoh, 1987; Goldberg et al., 2002). The choice for a temperature–stable sodium phosphate buffer resulted in a temperature sensitive, but pressure insensitive inactivation of ovomucoid (see OM-PhB8.0) under HPHT conditions (Grauwet et al., 2010b). Lack of pressure sensitivity at constant process temperature was attributed to a stronger pH drop of the phosphate buffer when a higher pressure level at a particular temperature was applied. Grauwet et al. (2010b) showed that the stability of ovomucoid increases when the pH of a buffer is lowered. In the context of extrinsic, isolated indicator development, the effect of a pH-shift, induced by a given pressure temperature change, on the kinetics of the indicator is included in the read-out evaluated after treatment and thus in its characterization in terms of kinetic properties. However, in this case, this raises no issues as the kinetics of the isolated system do not need to be translated to real food systems with different buffering capacities (Grauwet et al., 2010b). The choice of a pressure-stable buffer, with little effect of temperature was considered a logical starting point to develop an ovomucoid-based indicator system with a pressure sensitive inactivation. In this work, the use of a MES-NaOH buffer was opted (DpKa0/°C = 0.011; DV0 = 3.9 cm3 mol1; Bates, 1962; Kitamura and Itoh, 1987). In Fig. 2A, a clear effect of pressure on the residual trypsin-inhibitor activity could be observed when ovomucoid was treated under isobaric–isothermal conditions at 107 °C in a MESNaOH buffer of pHi 6.0. However, it needs to be verified if the presence of a pressure effect on the inactivation is not at the expense of a temperature effect. Indeed, in view of the applicability of the candidate pTTI for a temperature uniformity study, temperature should show a clear effect on the read-out of the system after treatment under constant, increased pressure. In Fig. 2B, a clear temperature effect on the inactivation of the ovomucoid system can be observed. Based on Fig. 2A and B, it can be concluded that by dissolving ovomucoid in a MES-NaOH buffer system (pHi 6.0), the ovomucoid system is characterized by a combined pressure temperature dependency of its inactivation, meeting the first objective.

Grauwet et al. (2009) developed a five-wise approach to develop extrinsic, isolated protein-based pTTIs for temperature uniformity mapping in HP vessels. The results reported below summarizing the optimization of the prototype ovomucoid-based sensor described by Grauwet et al. (2010b) will be described using this step-wise approach. In a first step, prerequisites a candidate indicator needs to have, are verified (e.g. food-grade, commercial availability, etc.). Since these prerequisites were already evaluated for an ovomucoid-based sensor by Grauwet et al. (2010b), the discussion of step 1 will not be repeated in this manuscript.

3.1.2. Effect of pHi The second objective of the solvent engineering study was to shift the ovomucoid inactivation window in the HPHT processing window relevant for commercial sterilization. The potential of the pHi of the selected buffer system to increase or decrease the ovomucoid stability under HPHT conditions was reported previously (Grauwet et al., 2010b). However, for each buffer type chosen, only a particular pHi-range can be studied in order to preserve the buffering capacities. In case of a MES-NaOH buffer, a pHi-range of pH 5–7 can be investigated.

where SSQregression, SSQtotal are the sum of squares of the estimated values and total, respectively, MSQresiduals is the mean squares of the residuals and x and p0 the number of observations and model parameters, respectively. All parameters estimated for the different modeling steps under isobaric–isothermal conditions were obtained through non-linear regression (SAS, version 9.2, USA). 2.5.2. Inactivation data obtained under dynamic pressure temperature conditions The feasibility of the selected kinetic models and their corresponding parameters obtained under isobaric–isothermal conditions to predict the read-out after a particular dynamic pressure temperature treatment was evaluated. Hereto, selected kinetic models and their corresponding parameters were inserted in Eq. (5) and the differential equation was solved by numerical integration of the registered pressure temperature profile (variable time interval <1.5 s; trapezium method was used), rendering the predicted read-out:

dðTIAÞ ¼ kðTÞ;ðpÞ ðTIAÞn dt

ð5Þ

with TIA representing the trypsin-inhibitor activity at treatment time t (min) and k(T),(p) the reaction rate constant (min1) under temperature and pressure, n the order of the reaction (n = 1 for first-order kinetics). Correlation between measured ovomucoid readings and predicted data was investigated. 2.5.3. Determination of significant differences between indicator readings evaluated after treatment at different positions in a temperature uniformity study Experimentally determined residual trypsin-inhibitor activities were assayed statistically using multiway ANOVA (SAS, version 9.2, USA). Significant differences among the pTTI read-outs were analyzed using the post hoc Tukey test. The significance level was set at a = 0.05.

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80

80

resTIA (%)

B 100

resTIA (%)

A 100

60 40 20

60 40 20

0

0

0

5

10

15

20

t iso(min)

0

5

10

15

20

tiso(min)

Fig. 2. Residual trypsin-inhibitor activity of 1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.0 after combined isobaric–isothermal treatments. (A) at 107 °C and 500 MPa (); 600 MPa (j); 700 MPa (j). (B) at 500 MPa and 107 °C (); 112.5 °C (); 118 °C (d).

It is not easy to define the HPHT window of commercial sterilization: the safety target attribute for HPHT is still not defined and in general, inactivation data of spores under HPHT are scarce. Some authors describe HPHT processing as a pressure-assisted thermal process (PATP). In this context, they advise pressure holding conditions at 121.1 °C for 3 min, since these isothermal processing conditions or equivalent processes lead to a 12Log-reduction of the safety target attribute for thermal sterilization at atmospheric pressure (C. botulinum; Holdsworth, 2009) (Barbosa-Canovas and Juliano, 2008; Juliano et al., 2009a). Consequently, for the selection of the PATP conditions, they disregard the potential synergism between pressure and temperature on the spore inactivation. However, different authors observed a synergistic effect of pressure and temperature under HPHT (500 MPa > p > 800 MPa) (Patazca et al., 2006; Ahn et al., 2007; Ramaswamy et al., 2009). In this work, it was aimed at shifting the inactivation window of ovomucoid in the processing range defined by pressures starting from 500 MPa, process temperatures starting from 105 °C applied during minimally 3 min. These processing conditions were expected to be relevant for HPHT conditions which result in fast spore inactivation. At the low impact end of an indicator application window, a candidate indicator should show minimal inactivation, so that different pressures and temperatures are reflected in different protein read-outs. At the high impact end of its application window, the selected system should show a minimal residual activity so the read-out is still detectable. In the following, isobaric–isothermal processing conditions of 500 MPa–107 °C and 700 MPa–119.5 °C were studied as relevant low and high impact ends for HPHT processing conditions, respectively. In Fig. 3A, the effect of the pHi-range of a MES-NaOH buffer on the isobaric–isothermal inactivation of ovomucoid under mild HPHT conditions (500 MPa–107 °C) is shown. As expected, the stability of ovomucoid was clearly pHi dependent in a MES-NaOH buffer (5.0–7.0): lower pHi resulted in a higher stability. In HPHT applications, short treatment times are intended. The stability of ovomucoid dissolved in a MES-NaOH buffer of pHi 5.0–5.5 was judged too high, since no clear inactivation occurred for this short treatment times. In addition, pHi of 7.0 was judged inappropriate because of too low stability: more than 40% of the indicator response was lost during the pressure build-up and equilibration phase (resTIA = 58% at tiso = 0 min). The latter candidate indicator system would reach the detection limit too fast. Subsequently, the stability of ovomucoid under intense processing conditions was studied (700 MPa–119.5 °C) in the pHi-range 6.0–6.7 (Fig. 3B). The stability of the ovomucoid system based on MES-NaOH-pHi 6.7 was insufficient under intense processing conditions: 75% of the indicator response was lost during the pressure build-up and equilibration phase (resTIA = 25% at tiso = 0 min). A still detectable read-out of ovomucoid was observed after process-

ing at intense conditions when ovomucoid was treated in a MESNaOH buffer in the pHi-range 6.0–6.5. 3.1.3. Selection of the solvent condition In Fig. 4A, the stability of ovomucoid dissolved in MES-NaOHpHi 6.2 after 3 min treatment under isobaric–isothermal conditions is shown. As ovomucoid (1 g/L) in this solvent condition (OMMB6.2) shows a clear combined pressure temperature inactivation in a processing domain relevant for HPHT sterilization, this protein-based candidate indicator was selected for further investigations. As mentioned above, a HPHT process consists of different processing steps. In this context, next to an appropriate stability of the candidate pTTI under HPHT conditions, an appropriate temperature stability of the indicator at atmospheric pressure is necessary. The third objective of this solvent engineering study was to improve the heat stability of the prototype ovomucoid system (OM-PhB8.0) at atmospheric pressure in the context of the preheating step before the actual HPHT treatment. Solid, low acid products with high moisture content show great potential to be treated under HPHT conditions. Assuming adiabatic conditions, starting from a Ti of 85–90 °C due to pressure build-up to 600– 700 MPa, the temperature of water can be increased to process temperatures around 120 °C (calculated based on using the database of the National Institute of Standards and Technology (NIST) and the International Association for Properties of Water and Steam (IAPWS)). Consequently, preheating temperatures of 85– 90 °C are very relevant for HPHT treatment of high moisture content food product. During a 15 min isothermal treatment of OMMB6.2 at 90 °C only 4% of residual trypsin-inhibitor activity was lost (Fig. 4B). To benefit maximally from the short come-up times due to compression heating in HPHT processing, the duration of the preheating phase should be minimized. Consequently, the heat stability of OM-MB6.2 at atmospheric was assessed sufficient. 3.2. Calibrating the indicator kinetics under isobaric–isothermal conditions To gain insight in the extent of the combined pressure temperature time dependency of the selected ovomucoid system, OMMB6.2, a detailed kinetic study was performed in the processing range of 107–119.5 °C; 500–700 MPa; 0–20 min and 90–130 °C; 0.1 MPa; 0–100 min (step 3 of indicator development; Grauwet et al., 2009). At all conditions tested, a clear combined effect of pressure, temperature and time on the read-out of OM-MB6.2 could be observed: inactivation rate increases as pressure and/or temperature increases. As an example, the residual trypsin-inhibitor activity as a function of isobaric–isothermal treatment time (tiso, min) at 0.1 MPa and 700 MPa are shown in Fig. 5A and B, respectively.

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T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

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60 40 20

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0

0

0

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tiso(min)

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tiso(min)

Fig. 3. Residual trypsin-inhibitor activity of 1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi  after combined isobaric–isothermal treatments (A) at mild processing conditions (500 MPa–107 °C): pHi 5.0 (N); pHi 5.5 (j); pHi 6.0 (); pHi 6.5 (d); pHi 7.0 () (B) at intense processing conditions (700 MPa–119.5 °C): pHi 6.0 (e); pHi 6.2 (+); pHi 6.5 (s); pHi 6.7 ().

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60 40 20

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550

600

650

700

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tiso(min)

Fig. 4. Residual trypsin-inhibitor activity of 1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.2 (OM-MB6.2) after combined isobaric–isothermal treatments. (A) as a function of pressure at 107 °C (); 110 °C (j); 112.5 °C (N); 116 °C (); 119.5 °C (d) (B) at atmospheric pressure and 90 °C (e).

3.2.1. Modeling the time-dependent inactivation The time-dependent inactivation of OM-MB6.2 was modeled by the first-order inactivation model (Fig. 5). Parameter estimation using non-linear regression was performed per temperature and pressure and the values obtained are listed in Table 1. Several observations were made: (i) both increasing the pressure at constant temperature and increasing the temperature at a given pressure leads to a higher inactivation rate constant, indicating a synergistic effect of both processing variables; (ii) depending on the processing conditions, some initial inactivation (resTIA0 < 100%) under HPHT conditions can be observed. The temperature sensitivity of this protein system suggests potential for temperature gradients detection through read-out evaluation of the indicators after treatment (observation i). The second observation makes it impossible to model both the time-dependent changes and the pressure temperature dependent changes in the rate constant in one step, unless the resTIA0 can be adequately pre-

dicted by a model in function of pressure and temperature. For each pressure temperature combination, the corresponding goodness-of-fit is expressed by R2adj (Eq. (3)). The RMSE-values (Eq. (4)) varied from 1.2 to 3.8. In Fig. 6, the graphical evaluation of this first modeling step is shown for all inactivation data obtained. The scatter and lag plots did not revealed specific trend. High correlation between measured and estimated values could be observed in the parity plot (R2 = 0.976). In the normal probability plot, no significant deviations from the bisector could be detected ðR2normal probability ¼ 0:996Þ. 3.2.2. Pressure temperature dependency of the reaction rate constant In a second phase, the pressure and/or temperature dependency of the inactivation rate constant was investigated. Neither the Arrhenius (1889), nor the Eyring equation (Eyring, 1946) could be used to satisfactorily describe the temperature and pressure dependency of the k-values, respectively, throughout the whole pressure temperature domain studied (data not shown). Conse-

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Fig. 5. Residual trypsin-inhibitor activity of ovomucoid (1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.2) after combined isobaric–isothermal treatments (A) at 0.1 MPa: 90 °C (); 100 °C (j); 105 °C (N); 110 °C (); 115 °C (s); 120 °C (d); 125 °C (+); 130 °C () (B) at 700 MPa: 107 °C (e); 110 °C (h); 112.5 (D); 116 (); 119.5 (+). Solid lines represent the first-order model fit.

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T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47 Table 1 Parameters predicted (± approximate standard error) based on first-order inactivation model for isobaric–isothermal inactivation of 1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.2 (OM-MB6.2): resTIA0, residual trypsin-inhibitor activity (%) at tiso = 0 min; k (min1), first-order inactivation rate constant. R2adj according to Eq. (3). p (MPa)

T (°C)

resTIA0 (%)

k (min1)

R2adj

0.1

90 100 105 110 115 120 125 130

102.4 ± 2.6 100.4 ± 2.2 102.8 ± 2.6 100.4 ± 4.6 100.9 ± 6.2 100.1 ± 4.0 102.4 ± 3.4 101.3 ± 3.4

0.005 ± 0.001 0.017 ± 0.001 0.022 ± 0.002 0.048 ± 0.005 0.053 ± 0.007 0.089 ± 0.008 0.139 ± 0.011 0.216 ± 0.015

0.948 0.983 0.985 0.976 0.962 0.983 0.983 0.991

500

107 110 112.5 116 119.5

102.3 ± 2.9 100.2 ± 4.9 99.5 ± 3.5 98.4 ± 5.8 97.6 ± 3.2

0.100 ± 0.007 0.118 ± 0.012 0.184 ± 0.017 0.233 ± 0.026 0.335 ± 0.022

0.969 0.952 0.990 0.972 0.990

600

107 110 112.5 116 119.5

102.3 ± 4.8 94.0 ± 3.5 90.8 ± 4.2 89.4 ± 3.7 84.0 ± 2.8

0.122 ± 0.012 0.142 ± 0.012 0.298 ± 0.026 0.334 ± 0.030 0.446 ± 0.028

0.977 0.966 0.982 0.983 0.988

700

107 110 112.5 116 119.5

95.5 ± 4.1 94.7 ± 4.2 92.5 ± 5.1 87.9 ± 1.8 79.7 ± 2.5

0.155 ± 0.016 0.215 ± 0.016 0.328 ± 0.039 0.416 ± 0.016 0.579 ± 0.040

0.976 0.973 0.969 0.989 0.991

quently, a single model describing both the pressure and temperature dependency was searched for. Several quadratic models in pressure and temperature were tested (for example, as used by Van Eylen et al. (2007), Buckow et al. (2009) and Katsaros et al. (2009)). For all data obtained under HP conditions, significance analysis of the different terms showed that only the temperature term and a combined pressure temperature term significantly contributed to the goodness-of-fit. In the literature, such a model equation has been termed the ‘combined Arrhenius–Eyring model equation’ (Eq. (2)). This model could adequately fit the combined

A

pressure temperature dependency of the inactivation rate constant (Fig. 7C) (R2 = 0.958). The scatter and lag plots did not reveal specific trends (Fig. 7A and B). In the normal probability plot, no significant deviations from the bisector could be detected ðR2normal probability ¼ 0:995Þ (Fig. 7D). The parameter estimates of the model, together with a summary of the statistics of regression are listed in Table 2. The estimated values for Ea and Va can be interpreted in the context of temperature or pressure dependency, respectively: the higher the absolute value, the higher is the process parameter sensitivity. Positive Ea- and negative Va-values demonstrate that inactivation rate increases as temperature and pressure are increased, respectively. In addition, negative Va-values show that the inactivation reaction is accompanied by a volume reduction. Comparing the Ea-values of the prototype system, OM-PhB8.0 (105.9 ± 8.2 kJ mol1; Grauwet et al., 2010b), to the new pTTI, OM-MB6.2 (127.7 ± 8.1 kJ mol1), it could be concluded that the development of a pressure sensitive sensor was not at the expense of the temperature sensitivity of ovomucoid.

3.3. Validating the kinetic model under dynamic pressure, temperature conditions As mentioned in the introduction, during the holding period, small differences in compression heating of different components in a HPHT reactor can result in temperature gradients and consequently dynamic temperature profiles. In addition, during the pressure build-up period, dynamic pressure temperature phases occur. In Section 3.2, it was shown that OM-MB6.2 can integrate the combined effect of pressure, temperature and time under isobaric–isothermal HPHT conditions. To be able to interpret the read-out of the ovomucoid system evaluated at different coordinates in a HPHT reactor based on the kinetic models and parameters obtained above, these integrating properties need to be verified under industrially relevant dynamic processing conditions (step 4 of indicator development). To that end, the residual trypsin-inhibitor activities measured after a set of treatments (x = 96) were compared to the residual trypsin-inhibitor activities calculated by both

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3 2 1 0 -15

-5 -1 -2 -3

ordered residuals

Fig. 6. (A) scatter plot of the residuals versus the predicted values; (B) lag plot of the residuals versus the residuals; (C) parity plot of the estimated versus the experimental values (R2 = 0.976); (D) normal probability plot ðR2normal probability ¼ 0:996Þ. All plots were made per p,T-combination for the first-order fitting of ovomucoid (1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.2) inactivation under isobaric–isothermal conditions.

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T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

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resTIA measured, first (%)

Fig. 7. (A) scatter plot of the residuals versus the predicted values; (B) lag plot of the residuals versus the residuals; (C) parity plot of the estimated versus the experimental values (R2 = 0.958); (D) normal probability plot ðR2normal probability ¼ 0:995Þ. All plots were made for the pressure temperature dependency of the first-order inactivation rate constant of ovomucoid (1 g/L ovomucoid in 0.1 M MES-NaOH buffer pHi 6.2) under isobaric–isothermal conditions predicted by the selected combined pressure temperature model (Eq. (2); Table 2).

Parameter

Estimate 1

krefpT (min ) Ea (kJ/mol) Va (cm3/mol) R2adjusted

0.128 ± 0.005 127.7 ± 8.1 9.05 ± 0.63 0.992

RMSE

0.008

inserting the dynamic pressure temperature profile, recorded at the actual position of the pTTI microtube, and Eq. (2), completed with the estimated parameters (Table 2), in Eq. (5). Dynamic pressure and temperature phases were observed during all treatment (data not shown). Neither the temperature profile recorded before pressure build-up nor that before 500 MPa was reached was used in the integration, as no inactivation of OM-MB6.2 was observed before that point. In Fig. 8, the correlation between measured and predicted read-outs is shown. The correlation was strong (R2 = 0.921), indicating the applicability of the kinetic model under a broad range of dynamic conditions relevant for industrial applications. 3.4. Construction of the isorate contour plot based on models and parameters valid under isobaric–isothermal and dynamic processing conditions

are connected. Several observations could be made: (i) the shape of the isorate contour plot of both indicator systems are not the same. OM-PhB 8.0 isorate contour plots are visualized as vertical straight lines in the pressure temperature window. OM-MB6.2 isorate contour plots are visualized as straight lines with a negative slope. As mentioned above, the sensitivity of both ovomucoidbased indicator systems towards the process parameter pressure is clearly different. However, increasing the temperature at constant pressure will result in both cases in an increase of the reaction rate constant and consequently faster inactivation. (ii) Processing conditions leading to the same inactivation rate constant are clearly different for both ovomucoid systems: the stability of OM-MB6.2 is clearly higher than OM-PhB8.0. In summary, based on the selection of one protein with potential to be used in the application window under consideration (Van

100

resTIA estimated, static para (%)

Table 2 Estimated parameters (± approximate standard error) for the model based on Eq. (2) describing the combined pressure temperature dependency of the first-order rate constant of inactivation of ovomucoid (1 g/L–MES-NaOH 0.1 M pHi 6.2), Tref = 110 °C; pref = 500 MPa.

80

60

40

20

0 0

As a basis for comparing both ovomucoid-based indicator systems, OM-PhB8.0 (Grauwet et al., 2010b) and OM-MB6.2, isorate contour plots were constructed based on the models and parameters valid under isobaric–isothermal and dynamic conditions in the domain of HPHT processing (Fig. 9). In an isorate contour plot, processing conditions leading to the same inactivation rate constant

20

40

60

80

10 0

resTIA measured, dyn (%) Fig. 8. Correlation between the residual trypsin-inhibitor activities estimated of ovomucoid (1 g/L–MES-NaOH 0.1 M pH 6.2) according Eqs. (2) and (5), using the parameters estimated from the isobaric–isothermal treatments Table 2 and the corresponding dynamic p,T-profile recorded and the experimentally determined activities after treatment (R2 = 0.921).

T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

der Plancken et al., 2005), solvent engineering enables the development of more than one protein system, each of them characterized by their own inactivation kinetics and own inactivation window, consequently their own application domain. 3.5. Temperature uniformity mapping in a HPHT reactor by read-out evaluation of a temperature sensitive ovomucoid system treated at different coordinates In a fifth step of the indicator development (Grauwet et al., 2009), the potential of the indicator developed to map temperature uniformity in a large-scale vessel was investigated. The idea to evaluate the process impact on a temperature sensitive indicator system to map the temperature uniformity in a HP reactor is not new (Denys et al., 2000; Van der Plancken et al., 2008). If the kinetics of the candidate pTTI are known, differences in indicator readings evaluated at different positions in the HP reactor, can be attributed to different numerical values of one or more process variables as a function of time. However, no experimental data are available in the literature in which such indicators are applied to map temperature uniformity in HPHT vessels. 3.5.1. Uniformity of the indicator’s read-outs in a HPHT vessel By positioning tubes filled with indicator solution OM-MB6.2 at six different vertical and horizontal coordinates in a HPHT reactor (Fig. 1), insight was gained in the temperature uniformity of a pilot-scale HPHT reactor. During the treatments performed, bottom heating was turned off to provoke temperature gradients and to evaluate the potential of the pTTI to detect temperature differences. As described under Section 2.3.2, pTTIs experienced the whole HPHT process: sample positioning; preheating at ambient pressure (4 min at 85 °C); HPTH treatment (Ti 85 °C; 600 MPa; 2 min versus 5 min); cooling at ambient pressure (4 min at 20 °C). It needs to be remarked that process intensities of the actual HPHT treatment in this pilot-scale HPHT unit are defined in terms of initial temperature (Ti) and not in terms of Tp as only control of the initial temperature of this HPHT unit is feasible. Each process intensity was tested in triplicate. Indicator readings evaluated at the same position for a particular process intensity were averaged over all treatments, each value provided with its standard error of treatment (x = 3). No trypsin-inhibitor activity was lost during process steps at ambient pressure (data not shown). Based on indicator readings

800

p (MPa)

700

600

500

400 90

100

110

120

130

T (°C) Fig. 9. pressure temperature combinations leading to a k-value of 0.145 min1 (dotted line) and 0.320 min1 (solid line) for inactivation of ovomucoid (OM-PhB8.0 (black); OM-MB6.2 (grey)) under isobaric–isothermal conditions simulated based on inactivation models and parameters valid under static and dynamic processing conditions (2-step regression).

45

evaluated after the whole HPHT process, several observations could be made (Fig. 10): (i) In all positions studied, a detectable read-out of OM-MB6.2 was evaluated or, in other words, the stability of this candidate pTTI developed was sufficient to be used under these intense HPHT conditions; (ii) For all positions, a significantly stronger inactivation of OM-MB6.2 was detected after treatment during 5 min (Fig. 10B) compared to after 2 min (Fig. 10A); (iii) For both process intensities studied (2 min versus 5 min), at a particular vertical zone (center (c) or wall (w)), a top-to-bottom stratification in read-out could be detected, measuring higher residual trypsin-inhibitor activities closer to the vessel bottom; (iv) For both processing intensities selected (2 min versus 5 min), at a particular horizontal zone (either top (t), middle (m) or bottom (b)) a center-to-wall stratification in read-out could be detected, measuring the higher residual trypsin-inhibitor activities closer to the vessel wall. 3.5.2. Mapping temperature uniformity in a HPHT vessel Based on the knowledge on the OM-MB6.2 inactivation kinetics (Sections 3.3 and 3.4), when pressure is uniform throughout the vessel and time is constant (batch process), differences in indicator readings evaluated at different treatment positions are due to different temperature histories. Consequently, although the protocol of this HPHT process includes the use of an isolating container and an isolating liner along the vessel wall, a non-uniform temperature distribution could be detected: the closer to the vessel bottom and the closer to the vessel wall, the lower impact temperatures histories could be detected in the HPHT vessel and vice versa. Under all processing conditions, the significant different and highest temperature zone could be detected at the vessel top in the center of the vessel. The significantly different and lowest temperature zone could be detected at the vessel bottom at the wall. In case of a positive temperature dependency of the kinetics of the targets attributes, these low and high temperature zones are important for process impact evaluation on safety and quality attributes, respectively. No significant different temperature histories were evaluated along the vessel wall. In addition, no significant differences could be observed between vessel wall and center at a specific height (top, middle, bottom) in the vessel, except at the top were small differences were observed. In vertical vessels, based on physical–mathematical simulations, center-wall stratification as well as top–bottom stratification in temperature have been predicted and attributed to conduction phenomena and/or free convection phenomena, respectively (Otero and Sanz, 2003; Ghani and Farid, 2007; Rauh et al., 2009). Indeed, since the compression heat of the vessel wall is almost zero, it becomes the cold region through which heat is being exchanged. Individual control of the temperature of the vessel wall on the one hand (Tp) and the temperature of sample and the incoming pressure on the other hand (Ti) has been put forward as a HPHT protocol to eliminate the vessel wall as cold region (end point strategy). However, in this case, the vessel wall can become the warm region. The potential of numerical simulation of temperature distributions in HP development has been proven in many literature reports (Denys et al., 2000; Hartmann and Delgado, 2002, 2003; Knoerzer et al., 2007; Otero et al., 2007; Rauh et al., 2009). However, these simulations require experimental information about the pressure and temperature dependent thermophysical properties (e.g. thermal conductivity, viscosity, density, compression heating properties, etc.) of the treatment media (e.g. transmitting medium, food product). The knowledge of the combined effect of temperature and pressure on these properties is far from complete (Otero and Sanz, 2003). In addition, numerical simulation demands high computational power and is case-dependent, requiring reevaluation if, for example, the load of the pressure vessel or the

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T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

B 100

100 80

AB C

B

AB

AB

A

60 40

resTIA (%)

resTIA (%)

A

80 60 DE

DE

40

D DE

DE F

20

20

0

0 top

middle

bottom

top

middle

bottom

Fig. 10. Experimentally determined residual trypsin-inhibitor activities of ovomucoid (1 g/L–0.1 M MES-NaOH pHi 6.2) after treatment at different positions in a HPHT vessel (Fig. 1) during 2 min (A) and 5 min (B) at Ti 85 °C and 600 MPa. In two different vertical sections (center (filled) versus wall (unfilled)), the effect of different distances from the vessel bottom were investigated (top; middle; bottom). Means indicated with the same particular letter are not significantly different. Means are provided with the standard error of treatment (x = 3).

vessel equipment design is changed. Extrinsic, isolated, proteinbased pTTIs can be used independently from the vessel load or the equipment design. In case of ovomucoid, the read-out can be quantified by an easy and fast spectrophotometrical measurement.

3.5.3. Critical reflection on the required temperature sensitivity of a HPHT indicator The potential of OM-MB6.2 to detect temperature differences under HPHT conditions has been shown Section 3.5.2. However, not all temperature differences will be translated in a significantly different indicator read-out. A pTTI is characterized by its temperature sensitivity (expressed by for example Ea (see Arrhenius equation and Eq. (2)); zT (see Thermal Death Time model)), the experimental error, the detection limit and its inactivation window. In food processing, there is only need to gain insight in those temperature differences in the relevant processing domain which will have an effect on the process impact on the food product. Theoretically, a distinction can be made between three cases: the temperature sensitivity of the indicator is (i) higher than, (ii) equal to, (iii) lower than the temperature sensitivity of the target attribute. The application of a pTTI is safe in the first two cases: temperature differences which will have an effect on the process impact on the target attribute will be indicated by process impact differences on the pTTI. In addition, in case (i), it is possible that temperature heterogeneities are detected by the indicator which will have no effect on the process impact uniformity of the target attribute. However, the lowest and highest temperature zones detected by the pTTI will be the lowest and highest impact zones on the target attribute in case of a positive temperature dependency. In case (iii), the potential of the indicator is restricted. By applying this indicator, different temperature zones may be indicated, however, within, for example, the indicated high temperature zone, a non-uniform process impact distribution of the target attribute can still occur. The effort necessary to apply the in situ method (i.e. direct evaluation of the process impact on the safety and quality attribute) at all different coordinates of the indicated low and high temperature zones in contrast to at all coordinates of the whole HP vessel will be totally different. In this line of thinking, although in case (iii), to some extent, one can still benefit from the indicators process impact evaluation. As the first objective of processing is ensuring food safety, the temperature sensitivity of spores will be compared to the temperature sensitivity of OM-MB6.2. Kinetic inactivation studies of spores under relevant HPHT conditions report zT-values of 21.5 °C; 27.2 °C and 35.36 °C at 600 MPa for the inactivation of spores of B. amyloliquefactiens (egg patty mince); G. stearothermophilus (deionized water) and G. stearothermophilus (ACES buffer pH 7.0), respectively (Patazca et al., 2006; Rajan et al., 2006;

Mathys et al., 2009). Using the Thermal Death Time model, a zT-value of OM-MB6.2 of 18.5 ± 1.0 °C at 600 MPa was calculated ðR2adj ¼ 0:953Þ. Consequently, the lowest and highest temperature zone mapped by OM-MB 6.2 would be the zones of lowest and highest impact on the spores, respectively (case i).

4. Conclusion By variation of the buffer type and the pHi in the context of solvent engineering, the first prototype ovomucoid-based pTTI (OMPhB8.0) to be used under HPHT conditions was optimized. This resulted in an indicator system (OM-MB6.2) characterized by combined pressure and temperature dependent first-order kinetics in the HPHT window relevant for commercial sterilization. Reaching commercial sterility under HPHT conditions implies a preheating period of the food product at atmospheric pressure (e.g. 85 °C 0.1 MPa), a HPHT treatment under intense HPHT conditions (e.g. Ti 85 °C–600 MPa–5 min) and a cooling phase (e.g. 20 °C– 0.1 MPa). By evaluation of the read-out of a temperature sensitive OM-MB6.2-based indicator systems treated at six different coordinates in a pilot-scale HPHT vessel, the potential of the pTTI for temperature uniformity mapping was verified. Low and high temperature zones could be detected. Based on this conclusion, the value of this study is dual: (i) In case of a positive temperature dependency of the target attributes, these low and high temperature zones will be important for direct process impact evaluation on safety and quality attributes, respectively. In this context, the indicator system developed can improve control of HPHT processes, which could aid process design and optimization. (ii) Next to the application of a pTTI in a temperature uniformity study, a pTTI can be applied for indirect process impact evaluation on a specific target attribute. For this type of application, the pressure temperature sensitivity and the relevant processing window of the pTTI and the target attribute should be equal. This work showed that both affecting the kinetics of an ovomucoid-based system, as well as shifting the inactivation window of the pTTI was feasible by the use of solvent engineering. Once the safety target attribute of HPHT is clearly defined and its kinetic data are available, the next challenge can be adjusting the inactivation characteristics of a candidate pTTI to the inactivation data of the safety target attribute to enable indirect process impact evaluation under HPHT.

Acknowledgements This work was financially supported by the Commission of the European Communities, Framework 6, Priority 5 ‘Food Quality and Safety’, Integrated Project NovelQ FP6-CT-2006-015710, the

T. Grauwet et al. / Journal of Food Engineering 105 (2011) 36–47

Research Fund of the Katholieke Universiteit Leuven and the Fund for Scientific Research Flanders (FWO).

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