g Technologies 8 (2007) 63–72www.elsevier.com/locate/ifset

Innovative Food Science and Emergin

A sterilisation Time–Temperature Integrator based on amylase from thehyperthermophilic organism Pyrococcus furiosus

G.S. Tucker a,⁎, H.M. Brown a, P.J. Fryer b, P.W. Cox b, F.L. Poole II c,H.-S. Lee c, M.W.W. Adams c

a Campden and Chorleywood Food Research Association, Chipping Campden, Glos., GL55 6LD, UKb Centre for Formulation Engineering, Department of Chemical Engineering, University of Birmingham, B15 2TT, UK

c Department of Biochemistry and Molecular Biology, University of Georgia, USA

Received 12 January 2006; accepted 7 July 2006

Abstract

A candidate Time–Temperature Integrator (TTI) which is potentially suitable for use in validation of sterilisation processes was identified andtested. The TTI was based on the highly thermostable amylase produced from the extracellular medium of a Pyrococcus furiosus fermentation:this organism grows at temperatures in the region of 100 °C. Kinetic properties for the amylase following inactivation by heat showed it to besuitable for use as a sterilisation TTI. Isothermal kinetic data at 121 °C and non-isothermal kinetic data over the range 121 to 131 °C weredetermined. A decimal reduction time (DT-value) at 121 °C of 24 min was calculated from isothermal tests and a range from 18.1 to 23.9 min fromnon-isothermal tests. A z-value of 10 °C was estimated from non-isothermal tests. Thus, sterilisation values (F0) estimated from this TTI would besimilar to F0-values representative of the destruction of Clostridium botulinum spores. Industrial measurements under non-isothermal conditionswere conducted in metal cans processed in an FMC reel and spiral cooker–cooler and a bar simulator, and also in plastic pouches processed in aLagarde steam-air retort.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Time–Temperature Integrator; TTI; Sterilisation value; Thermal processing; Canning

Industrial relevance: Many food processes, such as canning, are based upon thermal sterilisation of the food material. The development of areliable Time–Temperature Integrator for such a process would be industrially valuable by providing a simple way of validating such processes.This study demonstrates the feasibility of one such TTI.

1. Introduction

1.1. Industrial need for a sterilisation Time–TemperatureIntegrator

Thermal processing is probably the most important methodfor preserving food, and sterilisation processes such as canningare still widespread. The most heat-resistant pathogen that mightsurvive the thermal processing of low-acid foods is the spore-forming organism Clostridium botulinum. In practical terms, asterilisation process must reduce the probability of a singleC. botulinum spore surviving in a pack of low-acid product to

⁎ Corresponding author. Tel.: +44 1386 842035; fax: +44 1386 842100.E-mail address: g.tucker@campden.co.uk (G.S. Tucker).

1466-8564/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.ifset.2006.07.003

one in 1012. This is called a ‘botulinum cook’, and the standardprocess to achieve this level of spore reduction is equivalent to3 min at 121.1 °C, referred to as F0 3 (DoH, 1994; FDA, 2005).

Food manufacturers must prove that their products andprocesses are safe. Validation is usually carried out with tem-perature sensors, but this can be difficult for particulates thatmove within the processing system or for some packaging types.If temperature probes cannot be used, alternative approaches tovalidating microbiological process safety are required, such as:

• Microbiological methods, whereby cells or spores of a non-pathogenic organism, with similar temperature-induceddeath kinetics to the target pathogen, are embedded intoalginate beads (Brown, Ayres, Gaze, & Newman, 1984). Thebeads mimic food pieces in their thermal and physical

64 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

behaviour and so pass through the process with the food.Enumeration of the surviving organisms allows the logreduction and sterilisation value to be calculated.

• Simulated trials carried out in a laboratory where the heattransfer conditions of the process are replicated. Models suchas Ball (1923), Stumbo (1953), NumeriCAL (FMC Inc.,USA) or CTemp (Tucker, Noronha, & Heydon, 1996) predictthe required process conditions to achieve a desiredsterilisation value.

• Process models that predict, for example, the temperature–time history of the critical food particles as they travelthrough the heating, holding and cooling zones of the pro-cess (Heppell, 1985; Lee, Singh, & Larkin, 1990; McKenna& Tucker, 1991; Sastry, 1986).

Another option that is available to pasteurisation processes isthe use of time–temperature integrators (TTIs). A TTI can bedefined as a small measuring device that shows a time–tem-perature dependent irreversible change that mimics the changeof a target attribute when exposed to the same conditions. Inpractice, a TTI can be an enzyme, such as amylase or perox-idase, that denatures as it is heated in a buffer. If the reactionkinetics of the temperature-induced denaturation match those ofthe first order microbial death kinetics, the enzyme can be usedas a biochemical marker of a process. The development of TTIshas received considerable attention recently (see reviews byHendrickx et al., 1995; Maesmans et al., 1994).

Time–temperature integrators for pasteurisation processeshave been developed successfully (such as De Cordt, Hendrickx,Maesmans,&Tobback, 1992; Tucker, 1999; Van Loey, Hendrickx,DeCordt,Haentjens,&Tobback, 1996). It is nowpossible to use anamylase-based TTI for most commercial pasteurisation processes,from a few minutes at 70 °C up to many minutes at 95 °C (Tucker,Lambourne, Adams, & Lach, 2002). Recent TTI developments inpasteurisation have mostly used amylase from bacterial sourcessuch as Bacillus subtilis, amyloliquefaciens or licheniformis. Thefeasibility of extending its useable range upwards into sterilisationtemperatures was demonstrated by drying amylases to precisemoisture levels (De Cordt, Avila, Hendrickx, & Tobback, 1994;Guiavarc'h, 2003; Van Loey, Haentjens, Hendrickx, & Tobback,1997). Laboratory results were encouraging and showed thatdifferent levels of moisture content gave a range of heat stabilities.Thismethodmeasured the change in enthalpy for the dried amylasein a stainless steel pan within a differential scanning calorimeter.However, issues in sealing the pans from moisture ingress arise inindustrial canning plants (Tucker & Wolf, 2003). One furtherhurdle is the high pan density that prevents this method from beingused for flowing particulate systems. Therefore, a different methodis required for a sterilisation TTI.

The primary objective of the work reported here was to de-termine the feasibility of using amylase from an organism thatsurvives in extreme conditions as a candidate for a sterilisationTTI.

1.2. High temperature organisms

Previous work with TTIs has shown that amylases displaysuitable kinetic properties (Tucker, Cronje, & Lloyd, 2005;

Van Loey, Arthawan, Hendrickx, Haentjens, & Tobback, 1997;Van Loey, Haentjens, et al., 1997). Specifically, the measuredz-values for different amylases have been in the range from 9to 10 C°, ideal for bacterial spore destruction. Hence, anamylase was considered to provide the greatest chance offinding a TTI for use in sterilisation processes. The key was tolocate an organism that has evolved in high temperatureconditions and that produces amylase as it metabolises.

Microorganisms are known to exist in hostile environmentssuch as volcanic pools where they have adapted to hightemperature conditions and to chemical environments (Segereret al., 1993; Stetter, 1996). These ‘hyperthermophilic’ organ-isms represent a relatively new area for microbiological re-search and one with enormous potential for supply of heatstable enzymes (Sterner & Liebl, 2001). A number of bacteriacapable of growing at or above 100 °C have been isolatedfrom geothermic terrestrial and marine environments (Vieille& Zeikus, 2001). Among the many interesting featuresassociated with these bacteria are their ability to grow andcarry out biological functions at normally protein-denaturingtemperatures. Enzymes formed by these extremely thermo-philic and hyperthermophilic microorganisms are of greatinterest due to their thermostability and optimal activity at hightemperatures.

Amylases from hyperthermophilic organisms must be in-herently heat stable to hydrolyse starches in their favouredenvironmental conditions (Laderman, Davis et al., 1993;Leuschner & Antranikian, 1995; Niehaus, Bertoldo, Kahler,& Antranikian, 1999; Vieille & Zeikus, 2001). Hyperthermo-philic organisms that produce heat stable amylase includeClostridium thermohydrosulfuricum (Melasniemi, 1987;1988), Sulfolobus solfataricus (Worthington, Hoang, Perez-Pomares, & Blum, 2003), Desulfurococcus fermentans(Perevalova et al., 2005), Thermus thermophilus (Lioliou,Pantazaki, & Kyriakidis, 2004), Geobacillus thermoleovorans(Uma Maheswar Rao & Satyanarayana, 2004), Thermotogamaritima (Leuschner & Antranikian, 1995), Thermococcusceler (Blamey, Chiong, Lopez, & Smith, 1999), Fervidobac-terium pennavorans and Desulfurcoccus mucosus (Leuschner& Antranikian, 1995).

Pyrococcus furiosus was of great interest for the sterilisa-tion TTI because of the reported heat stability of its amylases(Koch, Zablowski, Spreinat, & Antranikian, 1990). Thearchaeon was isolated by Fiala and Stetter (1986) fromshallow thermal waters near Vulcano Island, Italy. P. furiosusis an obligate anaerobic, hyperthermophilic archaeon (orarchaebacterium). The motile coccus-shaped microbe, withabout 50 flagella at one end, is capable of growth on complexmedia with or without elemental sulphur. According to itsgenome sequence, P. furiosus contains at least five enzymesthat would be predicted to have amylase-type activity. Whenthe organism is grown on starch it produces an extracellularamylopullulanase (Brown & Kelly, 1993) and the recombinantform of an extracellular amylase has been characterized(Dong, Vieille, Savchenko, & Zeikus, 1997; Jorgensen,Vorgias, & Antranikian, 1997). In addition, an intracellularamylase has been purified from P. furiosus biomass

65G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

(Laderman, Davis et al., 1993) and the recombinant form wasalso obtained (Laderman, Asada et al., 1993). Furthermore,transcriptional analyses have shown that the gene encoding anextracellular amylase-type enzyme is up-regulated when theorganisms is grown on peptides rather than starch (Schut,Brehm, Datta, & Adams, 2003). P. furiosus is therefore apotentially rich source of amylolytic-type enzymes, althoughtheir exact function and the precise pathway by which starch ismetabolized is not clear.

Several groups have grown P. furiosus (Driskill, Kusy,Bauer, & Kelly, 1999; Koch et al., 1990; Ladermann, Daviset al., 1993, Ladermann, Asada et al., 1993; Savchenko,Vieille, Kang, & Zeikus, 2002; Verhagen, Menon, Schut, &Adams, 2001; Weinberg, Schut, Brehm, Datta, & Adams,2005). In one study, optimal growth and amylase production(more than 200 U l−1 after 8 h and 6.2×109 cells ml−1) wasobtained on a modified medium containing soluble starch andelemental sulphur, at 98 °C, pH 6.6 and under an 80/20 atmof H2/CO2 (Koch et al., 1990). Starch was randomly attackedby the amylase forming a mixture of various oligosaccharides.Eighty percent of the amylase was present in the culturesupernatant, which was typically the waste stream from afermentation.

P. furiosus amylases are also extremely thermostable. Ac-tivity has been measured over broad temperature (40–140 °C)and pH ranges (3.5–8.0). Optimum activity has been variouslyreported at 100 °C and pH 5 (Koch et al., 1990), between pH6.5–7.5 (Ladermann, Davis et al., 1993, Ladermann, Asadaet al., 1993) and at pH 5.6 (Brown, Costantino, & Kelly, 1990).No loss of activity was detected after 6 h of incubation at 90 °C(Koch et al., 1990), and at 120 °C, about 10% of the initialactivity was measured after 6 h. This equated to a decimalreduction time at 120 °C of 6 h (D120=6 h). To inactivate theenzyme completely, incubation had to be performed at 130 °Cfor at least 1 h. The material thus looks suitable for a sterilisationTTI. For successful use, the kinetics of thermal destruction ofamylase need to:

• Show sufficient heat stability for some of the active amylasestructure to remain after several minutes heating at 121.1 °C,characterised by the D-value. The commercial requirement forsterilisation processes is to achieve at least a process equivalentto 3 min at 121.1 °C. However, this is often increased to allowfor variability and to target spoilage microorganisms of higherheat resistance.

• Display a temperature sensitivity, characterised by a z-valueclose to 10 C°, which is used to represent the destruction ofC. botulinum spores.

2. Preparation of the candidate TTI

2.1. Production of P. furiosus amylase

This was undertaken at the University of Georgia, USA.P. furiosuswas grown on a rich medium containing yeast extractwith peptides as the primary carbon sources (Adams et al., 2001;Schut et al., 2003; Verhagen et al., 2001). The medium contained

seven separate components (a–g) prepared as separate sterilestock solutions and stored at 4 °C. Stock solutions were:

a) 5×salts solution, containing, per litre, 140 g of NaCl, 17.5 gof MgSO4 7H2O, 13.5 g of MgCl2 6H2O, 1.65 g of KCl,1.25 g of NH4Cl, and 0.70 g of CaCl2 2H2O

b) 100 mM Na2WO4 2H2O (10,000×, containing 33.0 g ofNa2WO4 2H2O per litre)

c) 1000× trace minerals solution, containing, per litre, 1 ml ofHCl (concentrated), 0.5 g of Na4EDTA, 2.0 g of FeCl3, 0.05 gof H3BO3, 0.05 g of ZnCl2, 0.03 g of CuCl2 2H2O, 0.05 g ofMnCl2 4H2O, 0.05 g of (NH4)2MoO4, 0.05 g of AlK(SO4)2H2O, 0.05 g of CoCl2 6H2O, and 0.05 g of NiCl2 6H2O

d) potassiumphosphate buffer, pH6.8 (1000×), containing 450mlof 1 M KH2PO4 (pH 4.3), to which 1 M K2HPO4 was addeduntil the solution reached pH 6.8 (approximately 550 ml)

e) 10% (wt/vol) yeast extract, consisting of 100 g of filter-sterilized yeast extract (DIFCO) per litre

f) 10% (wt/vol) casein hydrolysate, consisting of 100 g offilter-sterilized casein hydrolysate (enzymatic; U.S. Bio-chemicals) per litre

g) 50 g resazurin at 5 mg per ml.

The 5×salts solution and maltose were filter sterilized. Allother solutions were degassed and flushed with argon and storedat 4 °C. The reducing reagent consisted of cysteine HCl (0.5 g),Na2S (0.5 g) and NaHCO3 (1.0 g) per 500 ml adjusted to pH to6.8 with 1 M HCl. The solution was filter sterilized before use.

The peptides/S medium contained 0.5% (wt/vol) caseinhydrolysate (enzymatic), with sulphur added directly as a sus-pension to give a final concentration of 5 mg/ml.

The basal medium was composed of 1×base salts solutioncontaining, per litre, 200 ml of media (a), 0.1 ml of media(b), 1 ml of media (c), 0.05 ml of media (g), and 5 ml ofmedia (e). This was aseptically transferred into sterile serumvials (40 ml/100 ml bottle and/or 500 ml/1 L bottle), stopperedand autoclaved prior to adding the reducing agent. For theseed cultures, two 40 ml 1×base salt bottle were used. 0.2 mlof media (e) and 0.04 ml of media (d) were added to 40 ml1×base salt bottle.

For growth of the 1-litre culture, a fresh overnight culture ofP. furiosus was used to inoculate (2%, vol/vol) a 40 ml culturewhich was then grown overnight at 98 °C without stirring. Thiswas then used as an inoculum for one 500 ml culture containedin a one-litre flask, grown for 12 h at 98 °C to a cell density of∼ 2×108 cells/ml. Two 500 ml cultures were used to give a totalof one-litre culture for each. Cells were removed from theextracellular fraction by centrifugation at 10,000 ×g for 10 minat 4 °C. The supernatant was pink in colour because ofresazurin. Samples (2 ml) of the 1-litre culture were savedbefore and after removing the cells for activity assays. To the 1-litre supernatant, a total of 561 g of ammonium sulphate (80%)was added slowly over a 1-h period with stirring, and thesolution was allowed to stir for a further 16 h at 4 °C. Theprecipitated material was collected by centrifugation at10,000 ×g for 10 min. After decanting the supernatant, theprecipitate was sent at 4 °C by express mail to CCFRA.

66 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

On receipt of the precipitate at CCFRA, the ammoniumsulphate pellets were resuspended in an equivalent volume of50 mM ammonium bicarbonate buffer, pH 7.0. This wasdialysed against the same buffer to remove residual ammoniumsulphate. The dialysate was freeze-dried and the resultingfreeze-dried powder (FDP) used to prepare solutions for amy-lase assay. Table 1 gives the mass of FDP obtained, the proteincontent and amylase activity. The sample grown in a peptide-based medium gave 0.19 g FDP that was used for further tests todetermine its suitability as a sterilisation TTI.

2.2. Production of TTI tubes

One major advantage of a liquid TTI compared with a TTI inpowder form is the option of encapsulation within silicone TTItubes. These provide a TTI of neutral density in water with heattransfer characteristics, e.g. thermal conductivity and diffusivitysuitable for representing foods.

To make the TTI tubes, silicone tubing of 2.5 mm bore and0.5 mm wall (Altec; Alton, Hamphire) was cut into 10 mmlengths. One end was sealed by dipping it into uncured Sylgard170 elastomer (VWR International Ltd) and allowing capillaryaction to draw 2–3 mm of liquid up the tube. Heating the tube at70 °C for 30 min in an oven cured this end plug. A minimum of25 μl of the FDP solution was injected into each plugged tubeand uncured Sylgard 170 was drawn into the other end of thetube to form another 2–3 mm plug. The TTIs were then cured inan oven at 40 °C for approximately 40 min. Care was required toprevent drying of the solution or thermal damage to the amylase.

Once the FDP solution was encapsulated in the TTI tubes itwas ready for use. For trials reported here, TTI tubes wereattached to probes and placed within the food products. FilledTTI tubes were stored frozen in buffer until ready for use, whichincluded the time during transportation to and from theindustrial processing plants. Frozen storage can maintain highBacillus amylase activity for many months (Tucker et al.,2005), important for ensuring that the TTI has practical ap-plication. The two trials reported later were designed to chal-lenge this; one factory was located in East Anglia and the otherin the Scottish Highlands.

2.3. Assay methods

Continuous assays for amylase TTI systems for pasteurisa-tion had previously been conducted using reagent purchasedfrom Sigma or Randox (Tucker et al., 2005). ConventionalRandox assays were first conducted at 30 °C; however, amylasefrom P. furiosus had minimal activity at 30 °C and so the

Table 1Mass of FDP obtained from the Pyrococcus furiosus growth medium, thefreeze-dried protein contents and amylase activity

Growthmedia

Mass ofFDP (g)

Protein content (μgprotein/mg FDP)

Amylase activity (Δ600/min/20 μlof 1 mg protein/ml buffer)

Peptides 0.190 43.5 0.62

Buffer used with the FDP was 10 mM acetate, pH 5.0, 1 mM CaCl2.

standard test could not be used. An attempt was made to de-termine activity by adding 20 μl of FDP (15 mg resuspended perml of 10 mM acetate buffer, pH 5.0 containing 1 mM calciumchloride) to 1 ml of Randox amylase reagent at 90 °C (RandoxLaboratories, Catalogue number AY1580). Unfortunately, at90 °C the substrate precipitated from solution, hence this assaywas unsuitable for measurement of this thermostable amylase.The assay was repeated at 40–50 °C with only limited successbecause the low activity at these temperatures required lengthyincubation times.

A starch–iodine assay was then tested. Amylase activity wasmeasured by incubating at 92 °C a mixture of 20 μl of 1%soluble starch, 20 μl of 100 mM acetate buffer, pH 5.0 and 20 μlFDP (15 mg resuspended per ml of 10 mM acetate buffer, pH5.0 containing 1 mM calcium chloride). Incubation was for arange of times up to 15 min; no prior knowledge was availablefor appropriate incubation times. The reaction was stopped bythe addition of 1 ml of ice cold water and the colour developedby addition of 15 μl of an iodine solution (4% potassium iodideand 1.25% iodine solution). Colour changes from black toyellow were obtained as the amylase acted on the starch solu-tion; zero amylase activity gave a black colour whereas highactivity gave a yellow colour. Absorbance was read at 600 nmand plotted against incubation time. Activity (ΔA600 nm/min/20 μl sample) was calculated from the gradient of the line. Thisassay was chosen for the amylase from P. furiosus because ofthe need to operate at temperatures above 90 °C.

3. Determination of TTI kinetic parameters

3.1. Measurement of D-value: isothermal calibration

Traditionally, kinetic data are determined under a series ofisothermal experiments that give decimal reduction times (D-values) at each temperature. A log-linear relationship betweenthese D-values and temperature allows the z-value to be de-termined. Insufficient FDP was available to study multipletemperatures, so it was decided to measure its D-value only at121 °C to confirm whether the FDPwas of suitable heat stability.This required isothermal experiments to be conducted at 121 °Cusing the FDP in solution enclosed within glass capillary tubesthat were immersed in a well mixed glycerol bath at 121±0.2 °C.

Amylase activity was calculated from the change in absor-bance at 600 nm that corresponded to each point on the reactionrate curve. Tubes were incubated in an aluminium heater blockat 92 °C and the starch/iodine colour change determined foreach incubation time up to 15 min.

3.2. Measurement of D and z-values: non-isothermal calibration

Non-isothermal methods of obtaining D-and z-value datawere used as part of the industrial work. Methods for kinetic datadetermination followed closely those reported by variousresearch groups (De Cordt et al., 1992; Miles & Swartzel,1995; Van Loey, Arthawan, et al., 1997). TTIs were attached totemperature probes and the temperature–time profile, T(t)recorded. After the TTI had been through the process it was

Fig. 1. Time–temperature profile used to determine the non-isothermal kineticdata; (a) Lagarde trial 1 and (b) FMC Reel and Spiral trial 2.

67G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

assayed. Amylase activities from the TTIs and the temperaturesfrom the probes were converted sterilisation values using Eq. (1):

F ¼Z t

010

TðtÞ�Trefz ddt ¼ DTdlog

Ainitial

Afinal

� �ð1Þ

where,

F is the sterilisation value calculated at the referencetemperature (Tref), minutesAfinal is the final activityAinitial is the initial activityDT is the decimal reduction time at the reference temperature(Tref), minutesTref is the reference temperature, °Ct is the process time, minutesz is the temperature change required to effect a ten-foldchange in the DT value (C°)

Two variables define the F-values calculated with thesterilisation TTI and with temperature sensors: DT-value forreduction in amylase activity as measured with the sterilisationTTI and the z-value as calculated from measured times andtemperatures. A number of matching pairs of TTIs and in-tegrated temperature values gave pairs of calculated F-values.To obtain estimated values for DT and z the sum of the mini-mum absolute difference between matching paired values wasselected.

Two sets of experimental trials were carried out to provide awide range of F-values to challenge the measurement range ofthe TTI and thus estimate DT and z. It was important to measurea range of F-values calculated from a number of differentthermal processes, with all F-values measured at the end ofcooling. One unique pair of D121 and z-values was appropriatefor all of these sets of time–temperature data. To achieve a rangeof F-values, the data sets used different product heating rates aswell as different process temperatures between 121 and 131 °C.

3.2.1. Trial 1The first processing style used a commercial Lagarde steam-

air retort. Products were packaged in plastic pouches and glassjars. Various different thermal processes were given dependingon the product requirements to achieve commercial values forsterilisation. Different heating rates from the products allowedthe time–temperature data to differ in the rates of lethal rateaccumulation. Fig. 1(a) shows the different time-temperatureprofiles measured for these products. Values chosen for DT andz from trial 1 were used to estimate F-values from trial 2.

3.2.2. Trial 2The second processing style used a bar simulator for an FMC

reel and spiral cooker–cooler with cylindrical metal cans. In thissystem, fast axial rotation (FAR) occurred during parts of theprocess where the cans lost their contact with the reel. Thisresulted in extremely efficient heat transfer. The 610B barsimulator achieved this using FAR for one-third of the time ittook a can to travel around the reel. Water (0), 1 and 2% w/

w starch solutions were used to produce three different heatingrates for the product. Two different process temperatures wereused to provide data to challenge the kinetic calculations at 124and 131 °C. Fig. 1(b) shows the different time–temperatureprofiles measured for these products.

In both cases at least one sterilisation TTI was taped to the tipof a temperature sensor within the products. Tracksense loggers(Ellab UK Ltd, Kings Lynn) were used for the temperaturemeasurements. A common measuring position was assuredwithin a few millimetres for each matching pair of TTI andprobe.

4. Results

4.1. Measurement of DT by isothermal methods

Immersion of sealed glass capillary tubes in a well mixedglycerol bath at 121 °C was used to obtain the first D121-valuesfor the sterilisation TTI. These data are illustrated in Fig. 2(a)and (b), plotting the logarithm of the right side of Eq. (1) as afunction of immersion time. FDP concentration was 15 mg/mlbuffer. The data lies on good straight lines in Fig. 2(a) and(b), with D121-values calculated from the regression line as 18.1and 23.9 min respectively. Points in Fig. 2(a) were calculatedwith the initial activity estimated over the first 2 min ofincubation at 92 °C, whereas Fig. 2(b) used the first 5 min (seeFig. 3). This curve gave a period where the change in absorbancewith time was rapid; the gradient that represents the initial

Fig. 2. Plot of the ratio of activity before and after a given heating time at 121 °C for15 mg FDP/ml 10 mMAcetate buffer, pH 5.0 containing 1 mMCaCl2; (a)D-valueof 18.1min at 121 °C estimated from gradients taken over the first 2min incubationat 92 °C (b)D-value of 23.9min estimated from gradients taken over the first 5minincubation at 92 °C.

68 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

activity should be estimated from this period. It can be seen thatthe value for the initial activity depended to some extent on thetime period used to calculate the gradient. However, the effect ofinitial activities of 0.38 and 0.26 was less pronounced on theD121-values of 18.1 and 23.9 min respectively.

One limitation of using the FDP at 15 mg/ml buffer wasthe amount required to complete one measurement of either a D-value or of a series of activity rates for calculating a steriisationvalue. A standard TTI tube contained 25 μl of FDP solution,however, the starch/iodine assay required 20 μl to obtain a singlepoint on the reaction rate curve in Fig. 3. To obtain the full

Fig. 3. Change in absorbance at 600 nm for an unheated sample of FDP; at aconcentration of 15 mg FDP/ml 10 mMAcetate buffer, pH 5.0 containing 1 mMCaCl2. Initial FDP activity was estimated from the gradients of the reactioncurve over 2 and 5 min.

reaction rate, i.e. a gradient, there needed to be sufficient datapoints to define the curve; at least four incubation times werechosen. This equated to 100 μl of TTI solution, either in one TTIlarge tube or in four individual TTI tubes grouped together.Neither of these options was considered practical for industrialexperimentation. Estimation of a singleF-value required 4×20 μlfor the initial activity calculation (Ainitial) and 4×20 μl for the finalactivity calculation (Afinal). Thus, the method for gradientestimation was adjusted to maximise the number of kineticexperiments that could be done with only 190 mg of FDP.

An alternative method was investigated in which a higherconcentration of FDP was used (25 mg/ml), and the TTI solutiondiluted (5 mg/ml) before the assay was conducted. This allowedthe four replicates to be produced from the one sample and so fourpoints were obtained for calculating the gradient. Fig. 4 shows theplot of logarithm of activity ratio (initial activity divided by finalactivity) as a function of heating time. Each of the points in Fig. 4was determined with an effective FDP concentration of 5 mg/mlbuffer, considerably less than with the data in Fig. 2. The concernwas whether the reduced 5 mg/ml FDP concentration was highenough to measure amylase activities with sufficient accuracy. Itwas known from previous industrial trials with this sterilisationTTI that amylase activity decreased during storage. The D121-value was calculated from the regression line as 22.5 min, whichwas within the range of values from the experiments at 15 mgFDP/ml buffer. This suggested that the heat stability of thesterilisation TTIwas insensitive to FDP concentration in the range5 to 25 mg/ml buffer. Both were heated at 25 mg/ml, dilution wasthen to 15 or 5 mg/ml.

4.2. Measurement of DT and z by non-isothermal methods

D121.1 and z parameters were estimated from two industrialtrials. Each of the sterilisation TTIs was attached to a probe tipand contained approximately 25 μl of the FDP solution. Eachstarch–iodine assay required 20 μl from the TTIs, but it was notpossible to recover 20 μl from all of the TTIs because of lossesduring extraction. However, at least 15 μl was recovered fromeach TTI tube and an adjustment in activity was made for theTTIs where less than 20 μl was recovered.

As a result of diminishing quantities of FDP available fromthe U. Georgia broth, a more effective assay method was

Fig. 4. Plot of the ratio of activity before and after a given heating time at 121 °C;D-value of 22.5 min at 121 °C. 25 mg FDP /ml 10 mM Acetate buffer, pH 5.0containing 1 mM CaCl2 heated then diluted to 5 mg FDP/ml for assay.

Table 2(a)Sterilisation value data for trial 1; products in pouches processed in a Lagardesteam-air retort

CCFRA MF F(t−T) F(TTI) % Abs % Abs

Tube Code Min Min Diff Diff Diff Diff

1 1A 5.97 4.57 1.40 23.5 1.40 23.52 2A 4.18 3.57 0.61 14.6 0.61 14.63 3A 8.90 10.42 −1.52 −17.1 1.52 17.111 3B 9.37 11.14 −1.77 −18.9 1.77 18.913 5B 8.44 8.45 −0.01 −0.1 0.01 0.1

Ave −0.26 0.39 1.06 14.83

D121.1 was 21.45 min and z was 9.95C°.

Table 2(b)Sterilisation value data for trial 2; products in cans processed in an FMC reel andspiral cooker–cooler

CCFRA Baxters F(t−T) F(TTI) % Abs % Abs

Tube Code Min Min Diff Diff Diff Diff

2A 1 6.50 4.87 1.63 25.1 1.63 25.12B 13A 2 5.32 4.02 1.30 24.4 1.30 24.43B 2 5.32 4.15 1.17 21.9 1.17 21.94A 3 4.67 3.28 1.39 29.7 1.39 29.74B 3 4.67 3.12 1.54 33.1 1.54 33.15A 1 8.53 9.18 −0.65 −7.6 0.65 7.65B 16A 2 28.61 16.24 12.37 43.2 12.37 43.26B 2 28.61 19.07 9.54 33.4 9.54 33.47A 3 3.54 3.77 −0.23 −6.5 0.23 6.57B 3

Ave 4.51 19.11 4.87 24.76

D121.1 21.45 min and z 9.95C° used for estimating F-values.

69G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

derived from previous experiences with samples incubated at92 °C. Information on the colour changes over the 15-minuteincubation period at 92 °C had shown that the first 5-minutes ofincubation was critical in determining the reaction rate.Incubation beyond 5-minutes was not necessary. To maximisethe data obtained in the industrial experiments, the decision wasmade to optimise the assay by working with only two points onthe reaction curve; a time zero point and one at 5 min incubationat 92 °C. This assumed linearity in the measured colour changebetween zero and 5 min of incubation.

Data from the work on isothermal kinetics indicated that thisassumption resulted in a small underestimation of reaction rates

Fig. 5. Graphical illustration of F(t−T) and F(TTI) for trial 1 calculated usingD121.1 of 21.45 min and z of 9.95C°.; (a) products in pouches processed in aLagarde steam-air retort. (b); products in cans processed in an FMC reel andspiral cooker–cooler.

for the control (unheated) samples because the high amylaseactivity resulted in rapid starch degradation in the first fewminutes of incubation. Evidence for this can be seen from thegradients in Fig. 3. Heated samples showed linearity in reactionrate over a longer time period. Therefore, a ratio of the initialrate divided by the final rate was likely to underestimate the logreduction in amylase activity. It was considered that the positivebenefits of using only one 25 μl TTI for the assays outweighedthe negative of a slight underestimate of log reduction in amy-lase activity. The procedure for obtaining F-values from theindustrial experiments used:

F ¼ DTdlogðC0 � C05Þ=5ðC0 � Ct5Þ=5

� �ð2Þ

where,

C0 is the reading at 600 nm for the unheated control sampleafter 0-minutes incubation at 92 °C,C05 is the reading at 600 nm for the unheated control sampleafter 5-minutes incubation at 92 °C,Ct5 is the reading at 600 nm for the heated sample after 5-minutes incubation at 92 °C,

The advantage of non-isothermal TTI calibration is that itrepresents the behaviour of foods during thermal processing.Kinetic data (i.e. D and z) were evaluated with a series ofcoupled equations within an Excel workbook. The parametersused to determine values for D121.1 and z were the differencesbetween F-values calculated from the t–T data (referred to as F(t−T)) and from the TTI data (referred to as F(TTI)). Eq. (1)shows that calculations for F(t−T) require the z-value as theinput kinetic parameter, whereas those for F(TTI) require theD-value. Hence it was possible to estimate optimal values forthe D121.1 and z.

For trial 1, the minimum value for the average percentageabsolute difference between F(t−T) and F(TTI) was estimated

70 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

for D121.1 of 21.45 min and a z of 9.95 C°. Errors in D121.1 werelikely to be ±2 min and ±0.5 C° for z. Insufficient FDPwas available to permit a full statistical analysis of results andso estimation of errors was subjective. Two decimal places werecarried forward to trial 2 to maintain maximum accuracyfor intermediate calculations. Agreement between F(t−T) and F(TTI) for each of the paired valueswaswithin 1.5 units ofF-value,i.e. minutes. This was considered to be an acceptable level of errorwhen measuring F-values in the industrial range 3 to 15 min.

Table 2(a) and Fig. 5(a) show the data for trial 1. The best fit-line between paired values of F(t−T) and F(TTI) was adjustedto go through the origin; this had a minimal effect on D121.1 andz. It was likely that the minimum measurement for this steri-lisation TTI did not extend much below an F-value of 3 min, sothe lower region of the graph might be subject to a higher error.It will be more important for the sterilisation TTI that mea-surements of F-value are possible above the F 3 threshold forpublic health significance (DoH, 1994). Data from trial 2 wereevaluated using the same D121.1=21.45 min and z=9.95 C°to check on consistency. It can be seen from Table 2(b) and Fig.5(b) that there was good agreement between F(t−T) and F(TTI), although the highest F-values were 30–40% different.This level of accuracy was outside of that suggested by Pflug(1987) in which he justified a 20% difference. Improvement inthe accuracy will be achieved when more amylase becomesavailable for testing and the kinetic experiments can beconducted with replication. However, the accuracy reportedhere was acceptable for a novel TTI system in that itdemonstrated the potential for amylase from P. furiosus as asterilisation TTI.

It would be possible to achieve better agreement between F(t− T) and F(TTI) by adjusting the D and z-values inthe workbook for trial 2. However, with the exception of the F(t−T) value of 28 min, all other paired values were within 1.5 F-value units, and so the D and z-values from trial 1 were acceptedfor trial 2. Based on the values from trials 1 and 2, it was likely thatthe measurement range for this TTI was from F0 3 to 11 min.

5. Discussion of results

The data illustrated that an amylase from P. furiosus dis-played a thermal behaviour that was suitable for use as asterilisation TTI. D-values at 121 °C were measured between 18and 24 min for isothermal calibration and 24.5 min for non-isothermal calibration. Non-isothermal calibration for the z-value gave 10 C°, which was the same as the C. botulinum valueof 10 C°. F0-values measured with the sterilisation TTI wereaccurate to within 1.5 F0-value units of the F-values fromthermocouples over most of the measurement range. The ex-ception was for the single F0-value of 28 min where the TTIsystem gave a lower value. Obtaining high accuracy at high F0-values is not as important for process safety where the operatingregion is in the lower range towards F0 3. It may be that thesterilisation TTI cannot be used to measure more than one logreduction in amylase activity at the 25 mg/ml FDP concentra-tion. Operating ranges and further definition of accuracies needto be determined when more FDP is available.

Calibration of any measurement system is an essential require-ment in order to provide confidence that the values are correct andwithin a defined error band. Estimated errors displayed in Fig. 5(a)and (b) were ±10% on time–temperatureF-values and ±12.5% onTTI F-values. These errors were calculated from estimations ofinaccuracy with the measurement systems and variability with therelative experiments. Thermocouple temperature measurementswere assumed accurate to within ±0.5 °C under non-isothermalconditions, which converted to ±10% at, or close to, the 121.1 °Creference temperature. Estimated accuracies with TTI F-valueswere based on a change in D-value of ±3 min from the 24.5 mincalculated from the non-isothermal tests. This represented theupper and lower D-value limits from the non-isothermal cal-culations that gave acceptable agreement between F-values frompaired TTIs and probes. Further work will be needed to confirmwhether this is a realistic assessment.

F-values predicted using the calculated D121.1-value for thesterilisation TTIs were consistently within 1.5 F-value units ofthose from the time–temperature data for F-values in the range3.0 to 11.0 min. With most in-pack thermal processes operatingat around F0-values of 6 to 12 min, this is an acceptablemeasurement range and level of inaccuracy. Continuous thermalprocesses with particulates usually operate to substantiallyhigher F0-values because of the uncertainty involved with theirmeasurement. Thus, an error of ±1.5 min on a measured F-value in the region of 20–30 min would not be an issue.

6. Conclusions and future work

A candidate sterilisation TTI has been identified and testedbased on P. furiosus amylase. There are two main objectives forany thermal process measurement system: first to measure aprocess value so a food product can be produced safely, andsecond to optimise processes if the values are too high. Themeasurement range for this sterilisation TTI allowed both ofthese objectives to be realised.

Limitations in the quantity of FDP did not make it possible tocomplete all the testing appropriate for defining the limitationsof this TTI. Further experimental work is required in a numberof areas to address the questions that arose during the research,for example:

• It will be necessary to obtain larger quantities of FDP toenable further testing. Conditions used in the P. furiosusbatch fermentation may not have been optimised for amylaseproduction and may have resulted in detrimental by-products(e.g. proteases). Continuous fermentation could be used forgreater yields and consistency.

• The best conditions need to be determined for storing theFDP and of the filled sterilisation TTI tubes. This is im-portant to prevent loss in activity during transportation to/from industry trials.

• What level of amylase purification is required? The end pointfor work reported here was FDP since the intention was toinvestigate a candidate TTI. Reduction in activity was foundwhen the sterilisation TTIs were stored chilled, which wasthought to be caused by proteases acting on the amylase. Since

71G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

the FDP was not a pure amylase, other by-products of thefermentationwill be present, some ofwhichmay be detrimentalto the amylase.

• What variability should be expected for the sterilisation TTI?This TTI has many applications to industrial thermal processesand so it will be necessary to understand the accuracy of F-values estimated from the TTIs.

• How to guarantee long term supply of the FDP with repro-ducible heat stability properties. P. furiosus fermentation maynot be the best method to produce heat stable amylase. Thereare reports of the gene being expressed in bacteria such as E.coli or in moulds. Reports suggest that the amylase from an E.coli retains its heat stability but it has not been tested in thesame way as for a sterilisation TTI.

Acknowledgements

Funding from DEFRA LINK and the supporting industrialcompanies is gratefully acknowledged. AFM 194 (1 July 2003to 31 December 2006) involves CCFRA, University ofBirmingham, Marlow Foods, HJ Heinz Company Ltd., Green-core, Daniels Chilled Foods Ltd., Kerry Aptunion (UK) FruitPreparations, Masterfoods— a division of Mars UK Ltd., DeansFoods — Egg Products Division, Giusti Limited, SafetyEnvironmental Assurance Colworth, Baxters of Speyside Ltd.

Special thanks go to Johnston Pickles (Baxters of SpeysideLtd) and Steve Tearle (Masterfoods) for generating temperaturedata for the non-isothermal calibration.

Research conducted in the Adams laboratory was supportedin part by grants (BES-0317911 and MCB 0129841) from theUS National Science Foundation.

References

Adams, M. W. W., Holden, J. F., Menon, A. L., Schut, G. J., Grunden, A. M.,Hou, C., et al. (2001). Key role for sulfur in peptide metabolism and inregulation of three hydrogenases in the hyperthermophilic archaeon Pyro-coccus furiosus. Journal of Bacteriology, 183, 716−724.

Ball, C. O. (1923). Thermal process time for canned food. Bulletin of theNational Research Council, Washington, DC, Vol. 7. Part 1, Number 37.

Blamey, J., Chiong, M., Lopez, C., & Smith, E. (1999). Optimization of thegrowth conditions of the extremely thermophilic microorganisms Thermo-coccus celer and Pyrococcus woesei. Journal of Microbiological Methods, 38,169−175.

Brown, K. L., Ayres, C. A., Gaze, J. E., & Newman, M. E. (1984). Thermaldestruction of bacterial spores immobilised in food/alginate particles. FoodMicrobiology, 1, 187−198.

Brown, S. H., Costantino, H. R., & Kelly, R. M. (1990). Characterization ofamylolytic enzyme activities associated with the hyperthermophilicarchaebacterium Pyrococcus furiosus. Applied and Environmental Micro-biology, 56(7), 1985−1991.

Brown, S. H., & Kelly, R. M. (1993). Characterization of amylolytic enzymes,having both alpha-1,4 and alpha-1,6 hydrolytic activity, from thethermophilic archaea Pyrococcus furiosus and Thermococcus litoralis. Ap-plied and Environmental Microbiology, 59, 2614−2621.

Department of Health (1994). Guidelines for the safe production of heatpreserved foods. Norwich, England: Her Majesty's Stationery Office. ISBN0 11 321801 X.

De Cordt, S., Avila, I., Hendrickx, M., & Tobback, P. (1994). DSC and protein-based time−temperature integrators: case study of α-amylase stabilised bypolyols and/or sugar. Biotechnology and Bioengineering, 44, 859−865.

De Cordt, S., Hendrickx, M., Maesmans, G., & Tobback, P. (1992).Immobilised α-amylase from Bacillus licheniformis: a potential enzymictime–temperature integrator for thermal processing. International Journalof Food Science & Technology, 27, 661−673.

Dong, G., Vieille, C., Savchenko, A., & Zeikus, J. G. (1997). Cloning,sequencing, and expression of the gene encoding extracellular alpha-amylasefromPyrococcus furiosus and biochemical characterization of the recombinantenzyme. Applied Environmental Microbiology, 63, 3569−3576.

Driskill, L. E., Kusy, K., Bauer, M. W., & Kelly, R. M. (1999). Relationshipbetween glycosyl hydrolase inventory and growth physiology of thehyperthermophile Pyrococcus furiosus on carbohydrate-based media. Ap-plied and Environmental Microbiology, 65, 893−897.

FDA. (2005). Food and Drugs. Chapter 1— Food and Drug Administration.Department of Health and Human Services. Subchapter B— Food forHuman Consumption. Code of Federal Regulations, Title 21, Volume 2.Revised as of April 1, 2005 (21CFR113.3).

Fiala, G., & Stetter, K. O. (1986). Pyrococcus furiosus sp. nov. represents anovel genus of marine heterotrophic archaebacteria growing optimally at100 °C. Archives of Microbiology, 145, 56−61.

Guiavarc'h, Y. (2003). Development and use of enzymic time–temperatureintegrators for the assessment of thermal processes in terms of food safety.PhD Thesis No.570, Katholike Universiteit Leuven, Belgium.

Hendrickx, M., Maesmans, G., De Cordt, S., Noronha, J., Van Loey, A., &Tobback, P. (1995). Evaluation of the integrated time–temperature effect inthermal processing of foods. Critical Reviews in Food Science andNutrition, 35(3), 231−262.

Heppell, N. J. (1985). Measurement of the liquid–solid heat transfer coefficientduring continuous sterilisation of liquids containing particles. Proceedingsof IUFoST Symposium, Aseptic Processing and Packaging of Foods,Tylosand, Sweden (pp. 108).

Jorgensen, S., Vorgias, C. E., & Antranikian, G. (1997). Cloning,sequencing, characterization, and expression of an extracellular alpha-amylase from the hyperthermophilic archaeon Pyrococcus furiosus inEscherichia coli and Bacillus subtilis. Journal of Biological Chemistry,272, 16335−16342.

Koch, R., Zablowski, A., Spreinat, A., & Antranikian, G. (1990). Extremelythermostable amylolytic enzyme from the archaebacterium Pyrococcusfuriosus. FEMS Microbiology Letters, 71, 21−26.

Laderman, K. A., Asada, K., Uemori, T., Mukai, H., Taguchi, Y., Kato,I., et al. (1993). Alpha-amylase from the hyperthermophilic archaebac-terium Pyrococcus furiosus. Cloning and sequencing of the gene andexpression in Escherichia coli. Journal of Biological Chemistry, 268,24402−24407.

Laderman, K. A., Davis, B. R., Krutzsch, H. C., Lewis, M. S., Griko, Y. V., &Privalov, P. L. (1993). The purification and characterization of an extremelythermostable α-amylase from the hypertherophilic archaebacterium Pyro-coccus furiosus. Journal of Biological Chemistry, 32, 24394−24401.

Lee, J. H., Singh, R. K., & Larkin, J. W. (1990). Determination of lethality andprocessing time in a continuous sterilization system containing particulates.Journal of Food Engineering, 11, 67−92.

Leuschner, C., & Antranikian, G. (1995). Heat-stable enzymes from extremelythermophilic and hyperthermophilic microorganisms. World Journal ofMicrobiology and Biotechnology, 11, 95−114.

Lioliou, E. E., Pantazaki, A. A., & Kyriakidis, D. A. (2004). Thermusthermophilus genome analysis: benefits and implications. Microbial CellFactories, 3, 5.

Maesmans, G., Hendrickx, M., De Cordt, S., Van Loey, A., Noronha, J., &Tobback, P. (1994). Evaluation of process value distribution with timetemperature integrators. Food Research International, 27, 413−423.

McKenna, A. B., & Tucker, G. S. (1991). Computer modelling for the control ofparticulate sterilization under dynamic flow conditions. Food Control, 2,224−233.

Melasniemi, H. (1987). Characterization of alpha-amylase and pullulanaseactivities of Clostridium thermohydrosulfuricum. Evidence for a novelthermostable amylase. Biochemical Journal, 246, 193−197.

Melasniemi, H. (1988). Purification and some properties of the extracellularalpha-amylase-pullulanase produced by Clostridium thermohydrosulfuri-cum. Biochemical Journal, 250, 813−818.

72 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

Miles, J. J., & Swartzel, K. R. (1995). Evaluation of continuous thermalprocesses using thermocouple data and calibrating reactions. Journal ofFood Process Engineering, 18, 99−113.

Niehaus, F., Bertoldo, C., Kahler, M., & Antranikian, G. (1999). Extremophilesas a source of novel enzymes for industrial application. AppliedMicrobiology and Biotechnology, 51, 711−729.

Perevalova, A. A., Svetlichny, V. A., Kublanov, I. V., Chernyh, N. A.,Kostrikina, N. A., Tourova, T. P., et al. (2005). Desulfurococcusfermentans sp. nov., a novel hyperthermophilic archaeon from aKamchatka hot spring, and emended description of the genus Desulfur-ococcus. International Journal of Systematic and Evolutionary Microbi-ology, 55, 995−999.

Pflug, I. (1987). Factors important in determining the heat process value, FT, forlow-acid canned foods. Journal of Food Protection, 50, 528−533.

Sastry, S. K. (1986). Mathematical evaluation of process schedules for asepticprocessing of low acid foods containing discrete particulates. Journal ofFood Science, 51, 1323.

Savchenko, A., Vieille, C., Kang, S., & Zeikus, J. G. (2002). Pyrococcusfuriosus alpha-amylase is stabilized by calcium and zinc. Biochemistry, 41,6193−6201.

Schut, G. J., Brehm, S. D., Datta, S., & Adams, M. W. W. (2003). Whole-genome DNA microarray analysis of a hyperthermophile and an archaeon:Pyrococcus furiosus grown on carbohydrates or peptides. Journal ofBacteriology, 185, 3935−3947.

Segerer, A. H., Burggraf, S., Fiala, G., Huber, G., Huber, R., Pley, U., et al.(1993). Life in hot springs and hydrothermal vents. Origins of Life andEvolution of the Biosphere: the Journal of the International Society for theStudy of the Origin of Life, 23(1), 77−90.

Sterner, R., & Liebl, W. (2001). Thermophilic adaptation of proteins. CriticalReviews in Biochemistry and Molecular Biology, 36(1), 39−106.

Stetter, K. O. (1996). Hyperthermophiles in the history of life.Ciba FoundationSymposium, 202, 1−10 (discussion 11–18).

Stumbo, C. R. (1953). New procedures for evaluating processes for canned foodsin cylindrical containers. Food Technology, 7, 309−315.

Tucker, G.S. (1999). Application of time–temperature integrators for validationof pasteurisation processes. R&D Report No.77, CCFRA, ChippingCampden, Glos., GL55 6LD.

Tucker, G. S., Lambourne, T., Adams, J. B., & Lach, A. (2002). Application ofa biochemical time–temperature integrator to estimate pasteurisation valuesin continuous food processes. Innovative Food Science and EmergingTechnologies, 3, 165−174.

Tucker, G. S., Noronha, J. F., & Heydon, C. J. (1996). Experimental validationof mathematical procedures for the evaluation of thermal processes andprocess deviations during the sterilization of canned foods. Transactions ofthe IChemE. Food and Bioproducts Processing, Part C, 74, 140−148.

Tucker, G., Cronje, M. and Lloyd, E. (2005). Evaluation of a time–temperatureintegrator for mild pasteurisation processes. R&D Report No.215, CCFRA,Chipping Campden, Glos., GL55 6LD.

Tucker, G.S. and Wolf, D. (2003). Time–temperature integrators for foodprocess analysis, modelling and control. R&D Report No.177, CCFRA,Chipping Campden, Glos., GL55 6LD.

Uma Maheswar Rao, J. L., & Satyanarayana, T. (2004). Amelioration insecretion of hyperthermostable and Ca2+-independent α-amylase of Geo-bacillus thermoleovorans by some polyamines and their biosynthesisinhibitor methylglyoxal-bis-guanylhydrazone. Journal of Applied Microbi-ology, 97, 1015.

Van Loey, A. M., Arthawan, A., Hendrickx, M. E., Haentjens, T. H., &Tobback, P. P. (1997). The development and use of an α-amylase based time-temperature integrator to evaluate in-pack pasteurisation processes. Le-bensmittel-Wissenschaft und-Technologie, 30, 94−100.

Van Loey, A. M., Haentjens, T. H., Hendrickx, M., & Tobback, E. (1997). Thedevelopment of an enzymic time temperature integrator to assess the thermalefficacy of sterilization of low-acid canned foods. Food Biotechnology, 11(2), 147−168.

Van Loey, A. M., Hendrickx, M. E., De Cordt, S., Haentjens, T. H., &Tobback, P. P. (1996). Quantitative evaluation of thermal processes usingtime–temperature integrators. Trends in Food Science and Technology, 7,16−26.

Verhagen, M. F. J. M., Menon, A. L., Schut, G. J., & Adams, M. W. W. (2001).Pyrococcus furiosus: large-scale cultivation and enzyme purification.Methods in Enzymology, 330, 25−30.

Vieille, C., & Zeikus, G. (2001). Hyperthermophilic enzymes: sources, uses andmolecular mechanisms for thermostability. Microbiology and MolecularBiology Reviews, 65(1), 1−43.

Weinberg, M. V., Schut, G. J., Brehm, S., Datta, S., & Adams, M. W. W.(2005). Cold shock of a hyperthermophilic archaeon: Pyrococcus furiosusexhibits multiple responses to a suboptimal growth temperature with a keyrole for membrane-bound glycoproteins. Journal of Bacteriology, 187,336−348.

Worthington, P., Hoang, P., Perez-Pomares, F., & Blum, P. (2003). Targeteddisruption of the α-amylase gene in the hyperthermophilic archaeon Sulfo-lobus solfataricus. Journal of Bacteriology, 185, 482−488.