ink specialist and keen on the quality control

1. A sterilisation Time–Temperature Integrator based on amylase from the
hyperthermophilic 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, UK
b
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 and
tested. 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 be
suitable for use as a sterilisation TTI. Isothermal kinetic data at 121 °C and non-isothermal kinetic data over the range 121 to 131 °C were
determined. 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 from
non-isothermal tests. A z-value of 10 °C was estimated from non-isothermal tests. Thus, sterilisation values (F0) estimated from this TTI would be
similar to F0-values representative of the destruction of Clostridium botulinum spores. Industrial measurements under non-isothermal conditions
were conducted in metal cans processed in an FMC reel and spiral cooker–cooler and a bar simulator, and also in plastic pouches processed in a
Lagarde 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 a
reliable 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–Temperature
Integrator
Thermal processing is probably the most important method
for preserving food, and sterilisation processes such as canning
are still widespread. The most heat-resistant pathogen that might
survive the thermal processing of low-acid foods is the spore-
forming organism Clostridium botulinum. In practical terms, a
sterilisation process must reduce the probability of a single
C. botulinum spore surviving in a pack of low-acid product to
one in 1012
. This is called a ‘botulinum cook’, and the standard
process to achieve this level of spore reduction is equivalent to
3 min at 121.1 °C, referred to as F0 3 (DoH, 1994; FDA, 2005).
Food manufacturers must prove that their products and
processes are safe. Validation is usually carried out with tem-
perature sensors, but this can be difficult for particulates that
move within the processing system or for some packaging types.
If temperature probes cannot be used, alternative approaches to
validating microbiological process safety are required, such as:
• Microbiological methods, whereby cells or spores of a non-
pathogenic organism, with similar temperature-induced
death kinetics to the target pathogen, are embedded into
alginate beads (Brown, Ayres, Gaze, & Newman, 1984). The
beads mimic food pieces in their thermal and physical
Innovative Food Science and Emerging Technologies 8 (2007) 63–72
www.elsevier.com/locate/ifset
⁎ 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

2. behaviour and so pass through the process with the food.
Enumeration of the surviving organisms allows the log
reduction and sterilisation value to be calculated.
• Simulated trials carried out in a laboratory where the heat
transfer conditions of the process are replicated. Models such
as Ball (1923), Stumbo (1953), NumeriCAL (FMC Inc.,
USA) or CTemp (Tucker, Noronha, & Heydon, 1996) predict
the required process conditions to achieve a desired
sterilisation value.
• Process models that predict, for example, the temperature–
time history of the critical food particles as they travel
through 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 is
the use of time–temperature integrators (TTIs). A TTI can be
defined as a small measuring device that shows a time–tem-
perature dependent irreversible change that mimics the change
of a target attribute when exposed to the same conditions. In
practice, a TTI can be an enzyme, such as amylase or perox-
idase, that denatures as it is heated in a buffer. If the reaction
kinetics of the temperature-induced denaturation match those of
the first order microbial death kinetics, the enzyme can be used
as a biochemical marker of a process. The development of TTIs
has received considerable attention recently (see reviews by
Hendrickx et al., 1995; Maesmans et al., 1994).
Time–temperature integrators for pasteurisation processes
have been developed successfully (such as De Cordt, Hendrickx,
Maesmans, & Tobback, 1992; Tucker, 1999; Van Loey, Hendrickx,
De Cordt, Haentjens,& Tobback,1996).It is now possible to use an
amylase-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 in
pasteurisation have mostly used amylase from bacterial sources
such as Bacillus subtilis, amyloliquefaciens or licheniformis. The
feasibility of extending its useable range upwards into sterilisation
temperatures was demonstrated by drying amylases to precise
moisture levels (De Cordt, Avila, Hendrickx, & Tobback, 1994;
Guiavarc'h, 2003; Van Loey, Haentjens, Hendrickx, & Tobback,
1997). Laboratory results were encouraging and showed that
different levels of moisture content gave a range of heat stabilities.
This method measured the change in enthalpy for the dried amylase
in a stainless steel pan within a differential scanning calorimeter.
However, issues in sealing the pans from moisture ingress arise in
industrial canning plants (Tucker & Wolf, 2003). One further
hurdle is the high pan density that prevents this method from being
used for flowing particulate systems. Therefore, a different method
is 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 that
survives in extreme conditions as a candidate for a sterilisation TTI.
1.2. High temperature organisms
Previous work with TTIs has shown that amylases display
suitable kinetic properties (Tucker, Cronje, & Lloyd, 2005;
Van Loey, Arthawan, Hendrickx, Haentjens, & Tobback, 1997;
Van Loey, Haentjens, et al., 1997). Specifically, the measured
z-values for different amylases have been in the range from 9
to 10 C°, ideal for bacterial spore destruction. Hence, an
amylase was considered to provide the greatest chance of
finding a TTI for use in sterilisation processes. The key was to
locate an organism that has evolved in high temperature
conditions and that produces amylase as it metabolises.
Microorganisms are known to exist in hostile environments
such as volcanic pools where they have adapted to high
temperature conditions and to chemical environments (Segerer
et 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 heat
stable enzymes (Sterner & Liebl, 2001). A number of bacteria
capable of growing at or above 100 °C have been isolated
from geothermic terrestrial and marine environments (Vieille
& Zeikus, 2001). Among the many interesting features
associated with these bacteria are their ability to grow and
carry out biological functions at normally protein-denaturing
temperatures. Enzymes formed by these extremely thermo-
philic and hyperthermophilic microorganisms are of great
interest due to their thermostability and optimal activity at high
temperatures.
Amylases from hyperthermophilic organisms must be in-
herently heat stable to hydrolyse starches in their favoured
environmental 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 include
Clostridium 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), Thermotoga
maritima (Leuschner & Antranikian, 1995), Thermococcus
celer (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). The
archaeon was isolated by Fiala and Stetter (1986) from
shallow thermal waters near Vulcano Island, Italy. P. furiosus
is an obligate anaerobic, hyperthermophilic archaeon (or
archaebacterium). The motile coccus-shaped microbe, with
about 50 flagella at one end, is capable of growth on complex
media with or without elemental sulphur. According to its
genome sequence, P. furiosus contains at least five enzymes
that would be predicted to have amylase-type activity. When
the organism is grown on starch it produces an extracellular
amylopullulanase (Brown & Kelly, 1993) and the recombinant
form of an extracellular amylase has been characterized
(Dong, Vieille, Savchenko, & Zeikus, 1997; Jorgensen,
Vorgias, & Antranikian, 1997). In addition, an intracellular
amylase has been purified from P. furiosus biomass
64 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

3. (Laderman, Davis et al., 1993) and the recombinant form was
also obtained (Laderman, Asada et al., 1993). Furthermore,
transcriptional analyses have shown that the gene encoding an
extracellular amylase-type enzyme is up-regulated when the
organisms is grown on peptides rather than starch (Schut,
Brehm, Datta, & Adams, 2003). P. furiosus is therefore a
potentially rich source of amylolytic-type enzymes, although
their exact function and the precise pathway by which starch is
metabolized is not clear.
Several groups have grown P. furiosus (Driskill, Kusy,
Bauer, & Kelly, 1999; Koch et al., 1990; Ladermann, Davis
et 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
) was
obtained on a modified medium containing soluble starch and
elemental sulphur, at 98 °C, pH 6.6 and under an 80/20 atm
of H2/CO2 (Koch et al., 1990). Starch was randomly attacked
by the amylase forming a mixture of various oligosaccharides.
Eighty percent of the amylase was present in the culture
supernatant, which was typically the waste stream from a
fermentation.
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 variously
reported at 100 °C and pH 5 (Koch et al., 1990), between pH
6.5–7.5 (Ladermann, Davis et al., 1993, Ladermann, Asada
et 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 initial
activity was measured after 6 h. This equated to a decimal
reduction time at 120 °C of 6 h (D120 =6 h). To inactivate the
enzyme completely, incubation had to be performed at 130 °C
for at least 1 h. The material thus looks suitable for a sterilisation
TTI. For successful use, the kinetics of thermal destruction of
amylase need to:
• Show sufficient heat stability for some of the active amylase
structure to remain after several minutes heating at 121.1 °C,
characterised by the D-value. The commercial requirement for
sterilisation processes is to achieve at least a process equivalent
to 3 min at 121.1 °C. However, this is often increased to allow
for variability and to target spoilage microorganisms of higher
heat resistance.
• Display a temperature sensitivity, characterised by a z-value
close to 10 C°, which is used to represent the destruction of
C. 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. furiosus was grown on a rich medium containing yeast extract
with 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 sterile
stock solutions and stored at 4 °C. Stock solutions were:
a) 5×salts solution, containing, per litre, 140 g of NaCl, 17.5 g
of 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 of
Na2WO4 2H2O per litre)
c) 1000×trace minerals solution, containing, per litre, 1 ml of
HCl (concentrated), 0.5 g of Na4EDTA, 2.0 g of FeCl3, 0.05 g
of H3BO3, 0.05 g of ZnCl2, 0.03 g of CuCl2 2H2O, 0.05 g of
MnCl2 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) potassium phosphate buffer, pH 6.8 (1000×), containing450 ml
of 1 M KH2PO4 (pH 4.3), to which 1 M K2HPO4 was added
until 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 of
filter-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. All
other solutions were degassed and flushed with argon and stored
at 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 to
6.8 with 1 M HCl. The solution was filter sterilized before use.
The peptides/S medium contained 0.5% (wt/vol) casein
hydrolysate (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 solution
containing, 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 of
media (e). This was aseptically transferred into sterile serum
vials (40 ml/100 ml bottle and/or 500 ml/1 L bottle), stoppered
and autoclaved prior to adding the reducing agent. For the
seed cultures, two 40 ml 1×base salt bottle were used. 0.2 ml
of media (e) and 0.04 ml of media (d) were added to 40 ml
1×base salt bottle.
For growth of the 1-litre culture, a fresh overnight culture of
P. furiosus was used to inoculate (2%, vol/vol) a 40 ml culture
which was then grown overnight at 98 °C without stirring. This
was then used as an inoculum for one 500 ml culture contained
in 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 total
of one-litre culture for each. Cells were removed from the
extracellular fraction by centrifugation at 10,000 ×g for 10 min
at 4 °C. The supernatant was pink in colour because of
resazurin. Samples (2 ml) of the 1-litre culture were saved
before 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 the
solution was allowed to stir for a further 16 h at 4 °C. The
precipitated material was collected by centrifugation at
10,000 ×g for 10 min. After decanting the supernatant, the
precipitate was sent at 4 °C by express mail to CCFRA.
65G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

4. On receipt of the precipitate at CCFRA, the ammonium
sulphate pellets were resuspended in an equivalent volume of
50 mM ammonium bicarbonate buffer, pH 7.0. This was
dialysed against the same buffer to remove residual ammonium
sulphate. The dialysate was freeze-dried and the resulting
freeze-dried powder (FDP) used to prepare solutions for amy-
lase assay. Table 1 gives the mass of FDP obtained, the protein
content and amylase activity. The sample grown in a peptide-
based medium gave 0.19 g FDP that was used for further tests to
determine its suitability as a sterilisation TTI.
2.2. Production of TTI tubes
One major advantage of a liquid TTI compared with a TTI in
powder form is the option of encapsulation within silicone TTI
tubes. These provide a TTI of neutral density in water with heat
transfer characteristics, e.g. thermal conductivity and diffusivity
suitable for representing foods.
To make the TTI tubes, silicone tubing of 2.5 mm bore and
0.5 mm wall (Altec; Alton, Hamphire) was cut into 10 mm
lengths. One end was sealed by dipping it into uncured Sylgard
170 elastomer (VWR International Ltd) and allowing capillary
action to draw 2–3 mm of liquid up the tube. Heating the tube at
70 °C for 30 min in an oven cured this end plug. A minimum of
25 μl of the FDP solution was injected into each plugged tube
and uncured Sylgard 170 was drawn into the other end of the
tube to form another 2–3 mm plug. The TTIs were then cured in
an oven at 40 °C for approximately 40 min. Care was required to
prevent drying of the solution or thermal damage to the amylase.
Once the FDP solution was encapsulated in the TTI tubes it
was ready for use. For trials reported here, TTI tubes were
attached to probes and placed within the food products. Filled
TTI tubes were stored frozen in buffer until ready for use, which
included the time during transportation to and from the
industrial processing plants. Frozen storage can maintain high
Bacillus 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 other
in the Scottish Highlands.
2.3. Assay methods
Continuous assays for amylase TTI systems for pasteurisa-
tion had previously been conducted using reagent purchased
from Sigma or Randox (Tucker et al., 2005). Conventional
Randox assays were first conducted at 30 °C; however, amylase
from P. furiosus had minimal activity at 30 °C and so the
standard test could not be used. An attempt was made to de-
termine activity by adding 20 μl of FDP (15 mg resuspended per
ml of 10 mM acetate buffer, pH 5.0 containing 1 mM calcium
chloride) to 1 ml of Randox amylase reagent at 90 °C (Randox
Laboratories, Catalogue number AY1580). Unfortunately, at
90 °C the substrate precipitated from solution, hence this assay
was unsuitable for measurement of this thermostable amylase.
The assay was repeated at 40–50 °C with only limited success
because the low activity at these temperatures required lengthy
incubation times.
A starch–iodine assay was then tested. Amylase activity was
measured 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 μl
FDP (15 mg resuspended per ml of 10 mM acetate buffer, pH
5.0 containing 1 mM calcium chloride). Incubation was for a
range of times up to 15 min; no prior knowledge was available
for appropriate incubation times. The reaction was stopped by
the addition of 1 ml of ice cold water and the colour developed
by addition of 15 μl of an iodine solution (4% potassium iodide
and 1.25% iodine solution). Colour changes from black to
yellow were obtained as the amylase acted on the starch solu-
tion; zero amylase activity gave a black colour whereas high
activity gave a yellow colour. Absorbance was read at 600 nm
and plotted against incubation time. Activity (ΔA600 nm/min/
20 μl sample) was calculated from the gradient of the line. This
assay was chosen for the amylase from P. furiosus because of
the 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 of
isothermal experiments that give decimal reduction times (D-
values) at each temperature. A log-linear relationship between
these D-values and temperature allows the z-value to be de-
termined. Insufficient FDP was available to study multiple
temperatures, so it was decided to measure its D-value only at
121 °C to confirm whether the FDP was of suitable heat stability.
This required isothermal experiments to be conducted at 121 °C
using the FDP in solution enclosed within glass capillary tubes
that 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 reaction
rate curve. Tubes were incubated in an aluminium heater block
at 92 °C and the starch/iodine colour change determined for
each 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 data
were used as part of the industrial work. Methods for kinetic data
determination followed closely those reported by various
research groups (De Cordt et al., 1992; Miles & Swartzel,
1995; Van Loey, Arthawan, et al., 1997). TTIs were attached to
temperature probes and the temperature–time profile, T(t)
recorded. After the TTI had been through the process it was
Table 1
Mass of FDP obtained from the Pyrococcus furiosus growth medium, the
freeze-dried protein contents and amylase activity
Growth
media
Mass of
FDP (g)
Protein content (μg
protein/mg FDP)
Amylase activity (Δ600/min/20 μl
of 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.
66 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

5. assayed. Amylase activities from the TTIs and the temperatures
from the probes were converted sterilisation values using Eq. (1):
F ¼
Z t
0
10
TðtÞÀTref
z ddt ¼ DTdlog
Ainitial
Afinal
ð1Þ
where,
F is the sterilisation value calculated at the reference
temperature (Tref), minutes
Afinal is the final activity
Ainitial is the initial activity
DT is the decimal reduction time at the reference temperature
(Tref), minutes
Tref is the reference temperature, °C
t is the process time, minutes
z is the temperature change required to effect a ten-fold
change in the DT value (C°)
Two variables define the F-values calculated with the
sterilisation TTI and with temperature sensors: DT-value for
reduction in amylase activity as measured with the sterilisation
TTI and the z-value as calculated from measured times and
temperatures. 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 was
selected.
Two sets of experimental trials were carried out to provide a
wide range of F-values to challenge the measurement range of
the TTI and thus estimate DT and z. It was important to measure
a range of F-values calculated from a number of different
thermal processes, with all F-values measured at the end of
cooling. One unique pair of D121 and z-values was appropriate
for all of these sets of time–temperature data. To achieve a range
of F-values, the data sets used different product heating rates as
well as different process temperatures between 121 and 131 °C.
3.2.1. Trial 1
The first processing style used a commercial Lagarde steam-
air retort. Products were packaged in plastic pouches and glass
jars. Various different thermal processes were given depending
on the product requirements to achieve commercial values for
sterilisation. Different heating rates from the products allowed
the time–temperature data to differ in the rates of lethal rate
accumulation. Fig. 1(a) shows the different time-temperature
profiles measured for these products. Values chosen for DT and
z from trial 1 were used to estimate F-values from trial 2.
3.2.2. Trial 2
The second processing style used a bar simulator for an FMC
reel and spiral cooker–cooler with cylindrical metal cans. In this
system, fast axial rotation (FAR) occurred during parts of the
process where the cans lost their contact with the reel. This
resulted in extremely efficient heat transfer. The 610B bar
simulator achieved this using FAR for one-third of the time it
took a can to travel around the reel. Water (0), 1 and 2% w/
w starch solutions were used to produce three different heating
rates for the product. Two different process temperatures were
used to provide data to challenge the kinetic calculations at 124
and 131 °C. Fig. 1(b) shows the different time–temperature
profiles measured for these products.
In both cases at least one sterilisation TTI was taped to the tip
of a temperature sensor within the products. Tracksense loggers
(Ellab UK Ltd, Kings Lynn) were used for the temperature
measurements. A common measuring position was assured
within a few millimetres for each matching pair of TTI and
probe.
4. Results
4.1. Measurement of DT by isothermal methods
Immersion of sealed glass capillary tubes in a well mixed
glycerol bath at 121 °C was used to obtain the first D121-values
for the sterilisation TTI. These data are illustrated in Fig. 2(a)
and (b), plotting the logarithm of the right side of Eq. (1) as a
function of immersion time. FDP concentration was 15 mg/ml
buffer. The data lies on good straight lines in Fig. 2(a) and
(b), with D121-values calculated from the regression line as 18.1
and 23.9 min respectively. Points in Fig. 2(a) were calculated
with the initial activity estimated over the first 2 min of
incubation at 92 °C, whereas Fig. 2(b) used the first 5 min (see
Fig. 3). This curve gave a period where the change in absorbance
with time was rapid; the gradient that represents the initial
Fig. 1. Time–temperature profile used to determine the non-isothermal kinetic
data; (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

6. activity should be estimated from this period. It can be seen that
the value for the initial activity depended to some extent on the
time period used to calculate the gradient. However, the effect of
initial activities of 0.38 and 0.26 was less pronounced on the
D121-values of 18.1 and 23.9 min respectively.
One limitation of using the FDP at 15 mg/ml buffer was
the amount required to complete one measurement of either a D-
value or of a series of activity rates for calculating a steriisation
value. A standard TTI tube contained 25 μl of FDP solution,
however, the starch/iodine assay required 20 μl to obtain a single
point on the reaction rate curve in Fig. 3. To obtain the full
reaction rate, i.e. a gradient, there needed to be sufficient data
points to define the curve; at least four incubation times were
chosen. This equated to 100 μl of TTI solution, either in one TTI
large tube or in four individual TTI tubes grouped together.
Neither of these options was considered practical for industrial
experimentation. Estimation of a single F-value required 4×20 μl
for the initial activity calculation (Ainitial) and 4×20 μl for the final
activity calculation (Afinal). Thus, the method for gradient
estimation was adjusted to maximise the number of kinetic
experiments that could be done with only 190 mg of FDP.
An alternative method was investigated in which a higher
concentration of FDP was used (25 mg/ml), and the TTI solution
diluted (5 mg/ml) before the assay was conducted. This allowed
the four replicates to be produced from the one sample and so four
points were obtained for calculating the gradient. Fig. 4 shows the
plot of logarithm of activity ratio (initial activity divided by final
activity) as a function of heating time. Each of the points in Fig. 4
was determined with an effective FDP concentration of 5 mg/ml
buffer, considerably less than with the data in Fig. 2. The concern
was whether the reduced 5 mg/ml FDP concentration was high
enough to measure amylase activities with sufficient accuracy. It
was known from previous industrial trials with this sterilisation
TTI that amylase activity decreased during storage. The D121-
value was calculated from the regression line as 22.5 min, which
was within the range of values from the experiments at 15 mg
FDP/ml buffer. This suggested that the heat stability of the
sterilisation TTI was insensitive to FDP concentration in the range
5 to 25 mg/ml buffer. Both were heated at 25 mg/ml, dilution was
then 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 industrial
trials. Each of the sterilisation TTIs was attached to a probe tip
and contained approximately 25 μl of the FDP solution. Each
starch–iodine assay required 20 μl from the TTIs, but it was not
possible to recover 20 μl from all of the TTIs because of losses
during extraction. However, at least 15 μl was recovered from
each TTI tube and an adjustment in activity was made for the
TTIs where less than 20 μl was recovered.
As a result of diminishing quantities of FDP available from
the U. Georgia broth, a more effective assay method was
Fig. 3. Change in absorbance at 600 nm for an unheated sample of FDP; at a
concentration of 15 mg FDP/ml 10 mM Acetate buffer, pH 5.0 containing 1 mM
CaCl2. Initial FDP activity was estimated from the gradients of the reaction
curve over 2 and 5 min.
Fig. 2. Plot of the ratio of activity before and after a given heating time at 121 °C for
15 mg FDP/ml 10 mM Acetate buffer, pH 5.0 containing 1 mM CaCl2; (a) D-value
of 18.1 min at 121 °C estimated from gradients taken over the first 2 min incubation
at 92 °C (b) D-value of 23.9 min estimated from gradients taken over the first 5 min
incubation at 92 °C.
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 5.0
containing 1 mM CaCl2 heated then diluted to 5 mg FDP/ml for assay.
68 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

7. derived from previous experiences with samples incubated at
92 °C. Information on the colour changes over the 15-minute
incubation period at 92 °C had shown that the first 5-minutes of
incubation was critical in determining the reaction rate.
Incubation beyond 5-minutes was not necessary. To maximise
the data obtained in the industrial experiments, the decision was
made to optimise the assay by working with only two points on
the reaction curve; a time zero point and one at 5 min incubation
at 92 °C. This assumed linearity in the measured colour change
between zero and 5 min of incubation.
Data from the work on isothermal kinetics indicated that this
assumption resulted in a small underestimation of reaction rates
for the control (unheated) samples because the high amylase
activity resulted in rapid starch degradation in the first few
minutes of incubation. Evidence for this can be seen from the
gradients in Fig. 3. Heated samples showed linearity in reaction
rate over a longer time period. Therefore, a ratio of the initial
rate divided by the final rate was likely to underestimate the log
reduction in amylase activity. It was considered that the positive
benefits of using only one 25 μl TTI for the assays outweighed
the negative of a slight underestimate of log reduction in amy-
lase activity. The procedure for obtaining F-values from the
industrial experiments used:
F ¼ DTdlog
ðC0 À C05Þ=5
ðC0 À Ct5Þ=5
ð2Þ
where,
C0 is the reading at 600 nm for the unheated control sample
after 0-minutes incubation at 92 °C,
C05 is the reading at 600 nm for the unheated control sample
after 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 it
represents the behaviour of foods during thermal processing.
Kinetic data (i.e. D and z) were evaluated with a series of
coupled equations within an Excel workbook. The parameters
used to determine values for D121.1 and z were the differences
between 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 the
input kinetic parameter, whereas those for F(TTI) require the
D-value. Hence it was possible to estimate optimal values for
the D121.1 and z.
For trial 1, the minimum value for the average percentage
absolute difference between F(t−T) and F(TTI) was estimated
Table 2(a)
Sterilisation value data for trial 1; products in pouches processed in a Lagarde
steam-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 23.5
2 2A 4.18 3.57 0.61 14.6 0.61 14.6
3 3A 8.90 10.42 −1.52 −17.1 1.52 17.1
11 3B 9.37 11.14 −1.77 −18.9 1.77 18.9
13 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°.
Fig. 5. Graphical illustration of F(t−T) and F(TTI) for trial 1 calculated using
D121.1 of 21.45 min and z of 9.95C°.; (a) products in pouches processed in a
Lagarde steam-air retort. (b); products in cans processed in an FMC reel and
spiral cooker–cooler.
Table 2(b)
Sterilisation value data for trial 2; products in cans processed in an FMC reel and
spiral 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 25.1
2B 1
3A 2 5.32 4.02 1.30 24.4 1.30 24.4
3B 2 5.32 4.15 1.17 21.9 1.17 21.9
4A 3 4.67 3.28 1.39 29.7 1.39 29.7
4B 3 4.67 3.12 1.54 33.1 1.54 33.1
5A 1 8.53 9.18 −0.65 −7.6 0.65 7.6
5B 1
6A 2 28.61 16.24 12.37 43.2 12.37 43.2
6B 2 28.61 19.07 9.54 33.4 9.54 33.4
7A 3 3.54 3.77 −0.23 −6.5 0.23 6.5
7B 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

8. for D121.1 of 21.45 min and a z of 9.95 C°. Errors in D121.1 were
likely to be ±2 min and ±0.5 C° for z. Insufficient FDP
was available to permit a full statistical analysis of results and
so estimation of errors was subjective. Two decimal places were
carried forward to trial 2 to maintain maximum accuracy
for intermediate calculations. Agreement between F(t−T) and F
(TTI) for each of the paired values was within 1.5 units of F-value,
i.e. minutes. This was considered to be an acceptable level of error
when 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 adjusted
to go through the origin; this had a minimal effect on D121.1 and
z. It was likely that the minimum measurement for this steri-
lisation TTI did not extend much below an F-value of 3 min, so
the 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 for
public health significance (DoH, 1994). Data from trial 2 were
evaluated 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 in
the accuracy will be achieved when more amylase becomes
available for testing and the kinetic experiments can be
conducted with replication. However, the accuracy reported
here was acceptable for a novel TTI system in that it
demonstrated the potential for amylase from P. furiosus as a
sterilisation TTI.
It would be possible to achieve better agreement between F
(t − T) and F(TTI) by adjusting the D and z-values in
the 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 accepted
for trial 2. Based on the values from trials 1 and 2, it was likely that
the 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 a
sterilisation TTI. D-values at 121 °C were measured between 18
and 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 value
of 10 C°. F0-values measured with the sterilisation TTI were
accurate to within 1.5 F0-value units of the F-values from
thermocouples over most of the measurement range. The ex-
ception was for the single F0-value of 28 min where the TTI
system gave a lower value. Obtaining high accuracy at high F0-
values is not as important for process safety where the operating
region is in the lower range towards F0 3. It may be that the
sterilisation TTI cannot be used to measure more than one log
reduction in amylase activity at the 25 mg/ml FDP concentra-
tion. Operating ranges and further definition of accuracies need
to 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 and
within a defined error band. Estimated errors displayed in Fig. 5(a)
and (b) were ±10% on time–temperature F-values and ±12.5% on
TTI F-values. These errors were calculated from estimations of
inaccuracy with the measurement systems and variability with the
relative experiments. Thermocouple temperature measurements
were assumed accurate to within ±0.5 °C under non-isothermal
conditions, which converted to ±10% at, or close to, the 121.1 °C
reference temperature. Estimated accuracies with TTI F-values
were based on a change in D-value of ±3 min from the 24.5 min
calculated from the non-isothermal tests. This represented the
upper and lower D-value limits from the non-isothermal cal-
culations that gave acceptable agreement between F-values from
paired TTIs and probes. Further work will be needed to confirm
whether this is a realistic assessment.
F-values predicted using the calculated D121.1-value for the
sterilisation TTIs were consistently within 1.5 F-value units of
those from the time–temperature data for F-values in the range
3.0 to 11.0 min. With most in-pack thermal processes operating
at around F0-values of 6 to 12 min, this is an acceptable
measurement range and level of inaccuracy. Continuous thermal
processes with particulates usually operate to substantially
higher F0-values because of the uncertainty involved with their
measurement. 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 tested
based on P. furiosus amylase. There are two main objectives for
any thermal process measurement system: first to measure a
process value so a food product can be produced safely, and
second to optimise processes if the values are too high. The
measurement range for this sterilisation TTI allowed both of
these objectives to be realised.
Limitations in the quantity of FDP did not make it possible to
complete all the testing appropriate for defining the limitations
of this TTI. Further experimental work is required in a number
of areas to address the questions that arose during the research,
for example:
• It will be necessary to obtain larger quantities of FDP to
enable further testing. Conditions used in the P. furiosus
batch fermentation may not have been optimised for amylase
production and may have resulted in detrimental by-products
(e.g. proteases). Continuous fermentation could be used for
greater yields and consistency.
• The best conditions need to be determined for storing the
FDP 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 point
for work reported here was FDP since the intention was to
investigate a candidate TTI. Reduction in activity was found
when the sterilisation TTIs were stored chilled, which was
thought to be caused by proteases acting on the amylase. Since
70 G.S. Tucker et al. / Innovative Food Science and Emerging Technologies 8 (2007) 63–72

9. the FDP was not a pure amylase, other by-products of the
fermentation will be present, some of which may be detrimental
to the amylase.
• What variability should be expected for the sterilisation TTI?
This TTI has many applications to industrial thermal processes
and 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 may
not be the best method to produce heat stable amylase. There
are 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 the
same way as for a sterilisation TTI.
Acknowledgements
Funding from DEFRA LINK and the supporting industrial
companies is gratefully acknowledged. AFM 194 (1 July 2003
to 31 December 2006) involves CCFRA, University of
Birmingham, Marlow Foods, HJ Heinz Company Ltd., Green-
core, Daniels Chilled Foods Ltd., Kerry Aptunion (UK) Fruit
Preparations, Masterfoods— a division of Mars UK Ltd., Deans
Foods — Egg Products Division, Giusti Limited, Safety
Environmental Assurance Colworth, Baxters of Speyside Ltd.
Special thanks go to Johnston Pickles (Baxters of Speyside
Ltd) and Steve Tearle (Masterfoods) for generating temperature
data for the non-isothermal calibration.
Research conducted in the Adams laboratory was supported
in part by grants (BES-0317911 and MCB 0129841) from the
US National Science Foundation.
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