Journal of Food Engineering 155 (2015) 1–9

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Journal of Food Engineering

journal homepage: www.elsevier.com/locate/jfoodeng

Reducing the experimental effort in measuring D and z values for

microorganism inactivation kinetics
Gregory J. Fleischman ⇑
U.S. Food and Drug Administration, Institute for Food Safety and Technology, 6502 S. Archer Rd., Bedford Park, IL 60501, USA

a r t i c l e i n f o a b s t r a c t

Article history: For those microorganisms exhibiting log linear inactivation kinetics, the D and z values describing their
Received 15 May 2014 inactivation are typically measured in a series of isothermal experiments, each measuring log reductions
Received in revised form 17 December 2014 with time. This paper examines the mathematics of D and z measurement in order to show how both val-
Accepted 26 December 2014
ues could be obtained in fewer isothermal experiments than are traditionally performed. Additionally, a
Available online 22 January 2015
similar analysis of a non-isothermal experimental approach shows how even a minimum of 3 data points
in one experiment can be used to determine both parameters. This latter approach, though discussed pre-
Keywords:
viously in the literature, is given an analysis in this paper which reveals previously unrecognized details
Bacterial destruction
Thermal inactivation
of the method and its performance in extracting D and z values from experimental data.
D value Published by Elsevier Ltd.
z value

1. Introduction
The measurement of kinetic parameters describing the inactiva-
tion of microorganisms in various food environments is necessary
to help determine the efficacy of processes that seek to eliminate
them. The inactivation response is generally described by log linear
kinetics (Jay, 2000) where the rate of inactivation is proportional to
the number of surviving bacteria, leading to linear survival behav-
ior (when the survivor curve is plotted as the log of the survivors
against time). For microorganisms which exhibit linear survival
behavior, the kinetic parameters are the DT and z values. DT, the
decimal reduction time, is defined as the time needed to reduce
the viable microorganism count in a particular suspending med-
ium by a factor of 10 at a particular temperature, T. The z value
is defined as the change in temperature required to change DT by
a factor of 10. Together they are the basis for describing inactiva-
tion of microorganisms displaying log-linear kinetics as a function
of time and temperature. The basic procedure for obtaining these
two parameters is to determine the log reduction, log N0/N (where
N0 is the initial microorganism concentration and N is the surviving
microorganisms) as a function of time at a specific temperature,
which leads to DT, then obtaining values of DT at different temper-
atures which leads to z.
The common method for experimentally determining DT and z
employs a quick heating device that raises the temperature of a
bacterial suspension to a desired level, and then, at various times,
enumerating the survivors. Glass capillary tubes have had a long
history in this respect (Bigelow and Esty, 1920; Sognefest and
Benjamin, 1944). Other devices include the three neck flask (or
Woulff bottle) (National Canners Association, 1968), thermoresis-
tometer (Stumbo, 1948), thermal death time tank (Williams
et al., 1937), submerged coil (Cole and Jones, 1990) and plates
(Chung et al., 2008; Keller et al., 2012). The capillary tube itself
has also seen improvements over the years with the latest
described by Büchner et al. (2011). Regardless of the change in
design or sophistication of these apparatuses, the number of exper-
iments used to define DT and z has not changed. For example, if one
were to collect 4 data points, each involving 3 replicates, to obtain
DT, then evaluate DT at 3 other temperatures to get z, again with 3
replicates, a total of 48 enumerations of surviving microorganisms
need to be made. If z could be obtained using the same data col-
lected to calculate one value of DT, then only 12 enumerations
would be required—a significant savings in effort. Ironically, it is
the consistent intent to reduce come-up time that makes z difficult
to measure along with DT.
In this paper, it is shown how purposely extending the come-up
time allows the simultaneous determination of DT and z. This is
then taken to the extreme in which not only is the come-up time
extended but no constant temperature sampling occurs (i.e., all
samples are taken during the ‘‘come-up time’’), leading to a partic-
ularly straightforward simultaneous determination of DT and z.
2. Theory and calculations

⇑ Tel.: +1 708 924 0608 In what is to follow, the reader is referred to ‘Nomenclature’ for
E-mail address: gregory.fleischman@fda.hhs.gov parameter and variable definitions. Also, in anticipation of the

http://dx.doi.org/10.1016/j.jfoodeng.2014.12.023
0260-8774/Published by Elsevier Ltd.
2 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9

Nomenclature

D, DT decimal reduction time—time required to reduce micro- Tref reference temperature associated with a particular D
organism counts by 1 log at a particular T (min) value (°C)
K ramping rate (°C/min) Z temperature difference required to change DT by 1 log
N number of cells surviving thermal treatment (CFU/g) (°C)
N0 initial number of cells before thermal treatment (CFU/g) a see Eq. (5) or Eq. (7)
R log N0/N f dummy variable for integrating on time (min)
T temperature of the microorganism suspension (°C) s response time for first order heating (min)
t time (min) l mean
T0 initial temperature of the microorganism suspension rD standard deviation associated with evaluation of DT ref
(°C) (min)
Text heating medium temperature (°C) rR standard deviation associated with log reductions
Tlag the steady state lag between Text and the temperature (log CFU/g)
within a thermal death time device when Text increases rz standard deviation associated with evaluation of z (°C)
linearly (°C)

frequent use of log N0/N, R will be used instead to denote log Eq. (4) will be used to examine a method for simultaneous calcula-
reduction. tion of DT ref and z involving keeping time constant and varying tem-
The relationship between R and DT ref and z at constant temper- perature. For analyzing the standard method for calculating
ature is (Holdsworth and Simpson, 2008): DT ref and z, Tref can be used as Text, and therefore takes the place of
t Text in Eqs. (4) and (5):
R¼ 10ðTT ref Þ=z ð1Þ     a2  t  a3  t  
DT ref s t t
R¼  a es  1 þ e2s  1 þ e3s  1 þ 
For time-dependent temperature, the log reduction is given by
DT ref s 2  2! 3  3!
(Geankoplis, 2003). ð6Þ
Z t
1 where
R¼ 10ðTðfÞT ref Þ=z df ð2Þ
DT ref 0 T 0  T ref
a¼ ln 10 ð7Þ
Eq. (2) will be applied to two different functions of temperature ver- z
sus time. The first is the first-order response (exponentially asymp- These equations will be used to generate R versus t values for use in
totic temperature increase) associated with fast heating devices Monte Carlo simulations (Section 2.3) in Microsoft Excel. Therefore,
such as capillary tubes when plunged into a constant temperature a macro was written to sum the infinite series in Eqs. (4) and (6)
medium. It will therefore be applied to the standard isothermal (Appendix A.6). The two series are absolutely convergent by the
type of experiments to obtain DT and z. The second is a linearly comparison test (Johnson et al., 1974) using Eq. (11) in Section
increasing temperature which will form the basis for interpreting 2.3.1, which is shown to be absolutely convergent.
non-isothermal experiments to obtain DT and z.
2.2. Mathematical representation of bacterial reductions in non-
2.1. Mathematical representation of bacterial reductions in isothermal isothermal experiments
experiments
In this study, a non-isothermal method to measure bacterial
When first-order devices are placed in a constant temperature reductions uses a linearly ramped temperature, the form of which
environment, the temperature–time relationship in such devices is simply:
can be described with:
T ¼ Kt þ T 0 ð8Þ
T ext  T t
¼ es ð3Þ When Eq. (8) is substituted into Eq. (2), and the integration is car-
T ext  T 0
ried out, the log reduction is given as (see Appendix A.2):
where s depends on both the heating medium as well as the specific
z
device used (Coughanour and Koppel, 1965). These types of devices R¼ ð10ðTðtÞT ref Þ=z  10ðT 0 T ref Þ=z Þ ð9Þ
KDT ref ln 10
are designed to minimize internal temperature gradients by mini-
mizing the internal resistance to heat transfer compared to that in Eq. (9) can be simplified when examining the last two terms on the
the external heat transfer boundary layer. When Eq. (3) is substi- right and finding that, in most cases (discussed further under Sec-
tuted into Eq. (2), and the integration carried out (see Appendix tion 3.3) the latter is much smaller in comparison to the former
A.1 for details), the log reduction is given as: and therefore can be ignored. This allows the equation to be simpli-
  
s t t a2  2st  fied to:
R ¼ 10ðT ext T ref Þ=z  a es  1 þ e 1 !
DT ref s 2  2! TðtÞ  T ref z
 t   log R ¼ þ log ð10Þ
a3 z KDT ref ln 10
þ e3s  1 þ    ð4Þ
3  3!
This is a linear equation when the left hand side is plotted
where against T  Tref, with the inverse slope being z and the intercept
T 0  T ext the log term from which DT ref can be calculated.
a¼ ln 10 ð5Þ Similar to Eq. (10) was an equation obtained by Miles and
z
Mackey (1994), but using the Arrhenius kinetic approach where
 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9 3

the Arrhenius constant and activation energy replaced DT ref and z.
They derived their equation, having to make a similar assumption
as was needed to obtain Eq. (10) from Eq. (9), and applied it to
experimental data. This study supplements theirs by exploring
the potential limitations of that assumption, described in Section
3.3. Furthermore, this study also adds the potential effects of
experimental variations in log reductions, the effect of temperature
heating rates on the values of DT ref and z, and the effect of temper-
ature lag. The understanding of these effects is necessary for a
robust application of Eq. (10).
2.3. Monte Carlo simulation
To determine the robustness of the different methods for
obtaining DT ref and z, a Monte Carlo simulation was utilized. For
this purpose two sets of DT ref and z values, one for Salmonella Ente-
ritidis in chicken broth (Juneja et al., 2001) and the other for Salmo-
nella outbreak strains in peanut butter (Ma et al., 2009), were
obtained from the literature and are shown in Table 1.
In this equation, the exponential terms could be neglected because
the times were chosen such that t  s. Thus DT ref is calculated in
the usual way as the inverse slope of the inactivation line. However,
z is imbedded in the intercept via the parameter a (see Eq. (7)). To
back out z, a macro was written that would sum the series in brack-
ets (see Appendix A.7 for the Visual Basic code). By virtue of the ratio
test for infinite series, the series in Eq. (11) is absolutely convergent
(Johnson et al., 1974). Because a < 0, the series is alternating and,
therefore, accuracy is assessed by examining the nth term of the ser-
ies (Greenberg, 1978). However, because z is imbedded in each term
of the series it must be calculated implicitly. This means guessing a
value of z, recalculating the intercept, and seeing if this value
matches the ‘‘experimental’’ value. Excel’s GoalSeek routine is used
to adjust z to make the two intercept values match. A row containing
an experiment is then copied down to get 100 experiments. A macro
was used to apply GoalSeek to each row (see Appendix A.8 for the
Visual Basic code). Because z is naturally well bracketed, being
greater than zero and on the order of 50 °C or less, any guess in that
range was sufficient for GoalSeek to find a solution.

2.3.1. Simulations of isothermal experiments 2.3.2. Simulations of non-isothermal experiments

In isothermal experiments, Eq. (6) was used to generate values
of R for various times. Four time points were chosen in the linear
portion of the generated data (i.e., at times significantly beyond
the come-up time) to recalculate DT ref and z. The use of Eq. (6) also
requires a value of s, and three such values were chosen; 0.5 1.0,
and 1.5 min. To simulate variations in data, two reasonable stan-
dard deviations, rR = 0.1 and rR = 0.2, were chosen. A summary of
variables, parameters and their values for Monte Carlo simulation
of isothermal experiments is shown in Table 1. Fig. 1 shows the
layout of the sheet, the contents of which are described next.
An individual experiment comprised of four values of R for four
associated times occupied one row of an Excel spreadsheet, e.g.
row 10 in Fig. 1. These values were generated using the Excel func-
tion, NORM.INV, for generating values from a normal distribution
and imbedding the random number generator function RAND()
in it thus: NORM.INV(RAND(), l, r). By using a value of the gener-
ated R as l and one of the chosen values for r, a random number is
calculated that fits a normal distribution about the value of each R
in a row. A linear fit of these four points allows calculation of DT ref
and an intercept that leads to z. Both DT ref and the intercept are
placed in the same row.
The calculation of z proceeds according to a rearranged Eq. (6):
 
t s a2 a3
R¼ þ aþ þ þ 
DT ref DT ref 2  2! 3  3!
The same approach was followed as in Section 2.3.1, except that
both DT ref and z were calculated explicitly from the slope and as
part of the intercept of Eq. (10). The layout of the Excel spreadsheet
for non-isothermal experiments is shown in Fig. 2. Three values of
K were chosen; 0.5, 1.0 and 5.0 °C/min.
3. Results and discussion
Linear behavior in microorganism survivor curves is common.
Even in the so-called non-linear cases, survivor curves have multi-
ple linear portions such as in biphasic or triphasic behavior as well
as a linear portion with added features like shoulders, tails or both
(sigmoidal behavior). Xiong et al. (1999) was able to develop one
four-parameter equation that can be used to describe all the
behaviors except triphasic.
Regardless of the type of survivor behavior, deducing the corre-
sponding kinetic parameters with precision requires extensive
experimental effort. Gil et al. (2014) promote the use of optimal
experimental design to reduce this effort and increase precision
by focusing on replicates of a minimum number of optimally-cho-
sen points in the experimental domain. This minimum number is
usually equivalent to the number of kinetic parameters to be
deduced. However, they note that optimal experimental design is
ð11Þ not commonly utilized by microbiologists. Rather, in microbiology,
broad sampling of the experimental domain is relied upon to
increase precision of the parameter values. It is for this approach,

Table 1
Representative DT ref and z values and chosen values of s and K and the times used for each to generate R values to test the isothermal and non-isothermal methods.
Organism Salmonella Enteritidis Salmonella outbreak
PT 13 (Juneja et al., 2001) strains (Ma et al., 2009)
Medium Chicken broth Peanut butter
Tref (°C) 60 83
DT ref (min) 0.94 16
z (°C) 5.9 56
s (min) 0.5 1.0 1.5 10 20 30
Times (min) at each s used to obtain R 4 9 12 40 75 130
5 10 13 60 95 150
6 11 14 80 115 170
7 12 15 100 135 190
K (°C/min) 0.5 1.0 5.0 0.5 1.0 5.0
Times (min) at each K used to obtain R 62 33 7 80 50 18
66 34.5 7.5 120 65 22
68 36 8 140 80 24
71 37.5 8.25 150 95 26
4 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9

Fig. 1. The Excel spreadsheet layout for Monte Carlo simulations for the isothermal method. Rows 1–6 show the chosen parameters. Row 7 shows the chosen times. Row 8
holds the calculated values of R using Eq. (6). Row 9 holds the chosen standard deviation. Row 10 is a title line. Rows 11 downward each hold an ‘‘experiment’’ involving, from
left to right, ‘‘experimental’’ values of R obtained from the NORM.INV function, D fitted to the ‘‘experimental’’ values, z which will be determined, a calculation of a using z, the
intercept fitted to the ‘‘experimental’’ values and the intercept recalculated according to Eq. (11). The Excel function GoalSeek is used to adjust the value of z until the
recalculated intercept matches the ‘‘experimental’’ intercept.

Fig. 2. The Excel spreadsheet layout for Monte Carlo simulations for the non-isothermal method. Rows 1–5 show the chosen parameters. Row 6 shows the chosen times. Row
7 shows the temperatures associated with the chosen times per Eq. (8). Row 8 holds the calculated values of R using Eq. (9). Row 9 holds the standard deviation. Row 10 is a
title line. Rows 11 downward each hold an ‘‘experiment’’ involving, from left to right, ‘‘experimental’’ values of R obtained from the NORM.INV function, the log R values, a
calculation of z as the inverse slope of Eq. (10) using the T and log R values, and a calculation of DT ref as part of the intercept of the same equation.

when used to determine DT ref and z values for exclusively linear
survival behavior, that the methods below were developed.
3.1. The isothermal method
The isothermal method involves the measurement of DT ref and z
through data collected in constant temperature experiments. In
many cases, devices are used for this that contain the microorgan-
ism suspension and are submerged in a constant temperature bath.
They are designed to minimize come-up time as well as to mini-
mize internal temperature gradients so the temperature in the sus-
pension is uniform. Two common devices are capillary tubes for
heating liquid suspensions and plates (e.g., Chung et al., 2008, or
Keller et al., 2012) for pastes, or other semi-solids.
Ideally, such devices would instantaneously bring the suspen-
sion to the desired temperature. However, temperature traces
obtained from within these devices display a first order response
(Coughanour and Koppel, 1965), shown in Fig. 3 and described
by Eq. (3). The removal of these devices at various times in an
experiment to enumerate survivors occurs after the isothermal
part of the treatment has been reached. The value of DT ref is
obtained from the graph of R versus t as the inverse slope. The
slope, reflecting a change in survivor numbers over a change in
time, removes the effect of the non-isothermal portion of the curve
during which microorganisms inactivate, but at different tempera-
tures. Yet, it is in this part of the curve where the effect of z would
manifest itself. This is evident in Eq. (11) where z, imbedded in a
(Eq. (7)), is shown lurking in the intercept. The minimization of s
minimizes the effect of the intercept, but, again, the slope, and
therefore DT ref , is not affected by it. The only purpose, then, of a
device with small s is to reduce experimental time but there is
no fundamental need to make s small as long as the internal tem-
perature gradients within the device remain negligible. This opens
the possibility of using devices with purposefully large s to calcu-
late z in the same experiments that previously only resulted in
DT ref .
For devices with a first order response, Fig. 4 shows thermal
death times generated using measured DT ref and z of Salmonella
Enteritidis phage type 13 in chicken broth from Juneja et al.
(2001) (graph A) and for Salmonella outbreak strains in peanut
 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9 5

Eq. (11) in calculating DT ref and z in 100 sets of data generated using
different values for rR. The values of z and rz are the average values
from the 100 Monte Carlo experiments. The use of the standard
deviation (rR) to generate variations about a mean value of R is jus-
tified by the work of Cochrane (1950) who found that the experi-
mental variation around the log values of N can be described as a
near normal distribution, while their actual values are skewed
away from it.
Table 2 shows the results for Salmonella in chicken broth. Values
of rR for generating the variations were 0.1 and 0.2, with the
results for rR = 0 shown as well. The values for s are 0.5, 1.0 and
1.5 min. It can be seen that, in the rows, rz decreases as s increases.
Also, in the columns, rz increases as rR does. In terms of accuracy
of the value of z itself, increasing rR skews the value of z. At

Table 2
A comparison of z calculations from reductions generated for Salmonella Enteritidis in
chicken broth (Juneja et al., 2001) for different values of s and rR. The value of z and
rZ represents the average and standard deviation from all 100 Monte Carlo
experiments for the isothermal method.

Fig. 3. First order response of a device for heating bacterial suspensions in a rR z ± rz (°C) (actual, 5.9)
constant temperature water bath for different values of s. For this graph, T0 is 20 °C s (min)
and Text is 60 °C.
0.5 1.0 1.5
0.0 5.9 ± 0.02 5.9 ± 0.02 5.9 ± 0.01
0.1 6.3 ± 2.5 6.3 ± 1.9 6.3 ± 1.5
butter from Ma et al. (2009) (graph B). DT ref and z values could have 0.2 8.9 ± 7.1 7.7 ± 6.0 6.4 ± 2.8
been arbitrarily chosen but it was decided to use actual values to
demonstrate the span of thermal resistance found in practice. Four
values for s were chosen to generate the death time curves; 0, 0.5, Table 3
1.0, 1.5 min. The curves were generated using Eq. (6). The inclusion A comparison of z calculations from reductions generated for Salmonella outbreak
strains in peanut butter (Ma et al., 2009) for different values of s and r. The value of z
of the exponential functions in the infinite series of the equation
and rZ represents the average and standard deviation from all 100 Monte Carlo
allows the initial curvature of the thermal death time curves to experiments for the isothermal method.
be seen. Also shown are the lines drawn along the linear portion
rR z ± rz (°C) (actual, 56)
of each curve and extended to t = 0. These lines are described by
Eq. (11). s (min)
Fig. 4 indicates that an increasing value of s would enhance the 10 20 30
calculation of z by keeping the intercept away from the origin 0.0 59 ± 0.14 62 ± 0.09 60 ± 0.08
where the calculation of z cannot be made. This can be seen in 0.1 63 ± 16 63 ± 10 60 ± 10
the results shown in Tables 2 and 3. The tables summarize the 0.2 54 ± 18 61 ± 18 59 ± 16
Monte Carlo experiments used to determine the performance of

Fig. 4. Log reductions versus time calculated at four different values of s for a thermal death time device having a first order temperature response. Graph A shows curves for
values of DT ref and z corresponding to Salmonella Enteritidis phage type 13 in chicken broth, and graph B curves for Salmonella outbreak strains in peanut butter. Both sets of
values are shown in Table 1. The straight lines running along the linear portion of the curves corresponding to s = 0.5, 1.0 and 1.5 are meant to show the intercept of those
portions, which lead to the evaluation of z.
6 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9
rR = 0.2, only the greatest value of s keeps z near its stipulated
value.
Table 3 shows similar results for Salmonella in peanut butter.
Note, however, how great s has to be to utilize Eq. (11). The reason
is the much higher value of z necessitating longer times to capture
linear behavior. Therefore this method has a limit in that log reduc-
tions, though mathematically unconstrained, are practically con-
strained to experimentally viable values, usually around 5–6 due
to non-detectable limits in enumeration techniques. For large val-
ues of s, even these log reductions may not be within the linear
portion of the curve.
So far the discussion has centered on the calculation of z. The
average value of DT ref over all 100 Monte Carlo experiments for
both sets of data, regardless of s or r, resulted in values that devi-
ated less than 1% from the actual value (data not shown). This is
expected since the slope is independent of s.
3.2. The isochronal method
At this point it was decided to determine if another method
could be used where z would not be as sensitive to changes in R,
while retaining the simultaneous nature of the calculation of DT ref
and z. Eq. (4) provides some guidance here. This equation does
not assume that Text = Tref. Slightly rearranging Eq. (4) gives:
t s   t
R¼ 10ðT ext T ref Þ=z  10ðT ext T ref Þ=z a es  1
DT ref DT ref

a2  2 t
 Table 5, accuracy increased with K, but precision decreased. The
þ e s  1 þ 
2  2!
Eq. (12) is a complete description of R in that it includes any reduc-
tions occurring during the come-up time. If only the slope of this
equation is desired, as it is when only DT ref is needed, the following
equation is used:
t ðT ext T ref Þ=z
R¼ 10
DT ref
It would seem possible that Eq. (13) could be used to obtain DT ref
and z by taking the log of both sides:
t T ext  T ref
log R ¼ log þ ð14Þ
DT ref z
From Eq. (14), it is apparent that a set of experiments where t is
kept constant while varying Text, instead of the other way around,
would now yield both z, as the inverse slope, and DT ref , as part of
the intercept, when log R is plotted against Text. Eq. (12), however,
shows that Eq. (14) came about by ignoring the intercept. Therefore
Eq. (14) is not valid, except when s ¼ 0. This would mean a very fast
heating device is needed for measuring R. There would also be
another experimental difficulty in that samples, needing to be taken
at the same time but different temperatures, would require either
multiple constant temperature baths, or, if only one bath is avail-
able, multiple experiments to get multiple data points. This
approach does not appear to present any real savings in experimen-
tal effort over the traditional approach where Text is kept constant
and time is varied, wherein one experiment can yield multiple data
points. Because of this, no further analysis was performed on this
method.
3.3. The non-isothermal method
Instead, a method was found for the simultaneous calculation of
DT ref and z which retained the experimental simplicity of the iso-
thermal method and the mathematical simplicity of the isochronal
method. This method, which will be called the non-isothermal
method, collects reduction data while the suspending medium
undergoes linearly ramped heating.
By imposing a variable T that is linearly ramped in time, a sim-
ple solution to Eq. (2) can be found. By substituting Eq. (8) into Eq.
(2), Eq. (9) results. In most cases the second base 10 exponential
term is negligible, resulting in Eq. (10). It is evident that a plot of
log R versus T would give a line with a slope of 1/z and an intercept
from which DT ref is calculated.
This method was also studied using Monte Carlo simulations as
with the isothermal method, but with three different values of K
instead of s. Fig. 5 shows an example of the generated data. Of
the 100 experiments generated, four were chosen at random and
the data averaged at each of the four temperature points. Error bars
representing ± 2 standard deviations are also plotted. Table 4
shows the averaged results of all the Monte Carlo simulations for
Salmonella in chicken broth. In this case, however, decreasing K,
while showing decreased rz from K = 5 to K = 1, showed no further
decrease in rz in going to K = 0.5. More importantly, compare the
results of Tables 4 and 2, which is a comparison between the
non-isothermal and isothermal methods. For non-zero rR, the
non-isothermal method yielded values of z that were much more
accurate and precise (lower rz) than the isothermal method.
Table 5 shows the averaged results for all the Monte Carlo sim-
ulations for Salmonella in peanut butter. Regardless of rR, accuracy
and precision were approximately the same, showing the robust

nature of the method in encountering variations in data. However,
behavior of the values differed from what was seen in Table 4. In
ð12Þ accuracy, however, is due to the approximation where the second
base 10 exponential term of Eq. (9) is assumed small and ignored.
In the case of Salmonella in peanut butter, this term, though still
small, is not small enough to be ignored. The following equation
is therefore derived (see Appendix A.3) to include the influence
of the previously omitted exponential term:
!
TðtÞ  T ref 1 z
ð13Þ log R ¼  10Kt=z þ log ð15Þ
z ln 10 KDT ref ln 10
Temperatures in the middle term of the right hand side of Eq.
(15) were replaced with Kt using Eq. (8) and now it can be seen
why increasing K increases accuracy—the greater K, the smaller
the middle term. To keep this error term small, say on the order
of 1% or less, a minimum sampling time can be derived (see
Appendix A.4):
Fig. 5. A plot of log N0/N versus temperature to show an example of a fit to the
generated data from the Monte Carlo experiments for the non-isothermal method.
Four such experiments were chosen, with values averaged and the standard
deviations calculated. The plot shows error bars representing ± two standard
deviations, or about 95% of the variability, for each experimental point.
 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9 7

Table 4
A comparison of DT ref and z calculations from reductions generated for Salmonella
Enteritidis in chicken broth (Juneja et al., 2001) for different values of K and rR. The
values of z and rZ, and DT ref and rD represent the average and standard deviation from
all 100 Monte Carlo experiments for the non-isothermal method.
valid when the entire bacterial suspension experiences the same
lag. Therefore, this is not a correction for devices that allow temper-
ature gradients to exist within the microbial suspension.Lag also
affects the inequalities of Eqs. (16) and (17). They become:

rR z ± rz (°C) (actual, 5.9) 2z þ T lag

DT ref ± rD (°C) (actual, 0.94)
t> ð19Þ
K
K (°C/min1) and
0.5 1.0 5.0
z þ T lag
0.0 5.9 ± 0.0003 5.9 ± 0.0002 5.9 ± 0.0003 t> ð20Þ
K
0.94 ± 0.00005 0.94 ± 0.00002 0.94 ± 0.0001
0.1 5.9 ± 0.3 5.9 ± 0.2 5.8 ± 0.3
To determine when Tlag is important, the following equation is
0.94 ± 0.1 0.94 ± 0.02 0.94 ± 0.1 used (see Appendix A.5):
0.2 5.9 ± 0.6 5.9 ± 0.5 5.8 ± 0.7 DT ext
0.95 ± 0.1 0.94 ± 0.04 0.94 ± 0.1 T lag ¼ z log ð21Þ
DT 1
If a 5% or less deviation between D values is desired then the
ratio of the D values in Eq. (21) is 0.95 and, if z is 5.9 °C, then
Tlag < 0.13 °C. For z = 55, Tlag < 1.2 °C. It appears that even a small
Table 5 Tlag can affect the value of D. Fortunately, simply knowing the lag
A comparison of DT ref and z calculations from reductions generated for Salmonella
allows easy correction of D.
outbreak strains in peanut butter (Ma et al., 2009) for different values of K and rR. The
values of z and rZ, and DT ref and rD represent the average and standard deviation from A final note involves the work of Reichart (1979). In that study,
all 100 Monte Carlo experiments for the non-isothermal method. a non-isothermal approach was also taken to obtain D and z values
simultaneously from microorganism survivor curves. However, the
rR z ± rz (°C) (actual, 56)
DT ref ± rD (°C) (actual, 16) author did not attempt to maintain a linear increase in tempera-
ture and therefore did not obtain equations that could be fit to
K (°C/min1)
data. Rather, a graphical approach was taken where both the tem-
0.5 1.0 5.0 perature of the suspending medium and log reductions were plot-
0.0 49 ± 0.003 52 ± 0.03 54 ± 0.03 ted against time. D values were obtained by measuring the slope of
16 ± 0.001 17 ± 0.03 16 ± 0.016 the tangent line at particular time points along the survivor curve.
0.1 49 ± 3.1 52 ± 2.6 54 ± 2.9 The time associated with each such D value was then used on the
16 ± 0.5 17 ± 0.33 17 ± 1.7 graph of temperature versus time to obtain the temperature of the
0.2 50 ± 6.0 51 ± 5.5 55 ± 5.9 particular D value. This allows z to be calculated. The survivor
16 ± 1.0 17 ± 0.69 17 ± 3.8 curves used in the paper were smooth, but highly non-linear.
While this approach appears viable at first glance, it relies entirely
on the ability to measure the slope of a tangent line accurately at
individual time points to obtain D at those points. Even with very
z
t>2 ð16Þ accurate data, it is nearly impossible to obtain accuracy measuring
K such slopes graphically with a straight-edge. Numerical
Since variability of R is much greater than 1%, a 10% error may approaches exist, but all are advised against, primarily because
be tolerable, in which case: even with accurate data, numerical differentiation (which is what
z measuring the slope of a tangent line is) will magnify small dis-
t> ð17Þ crepancies (Carnahan et al., 1969; Kreyszig, 1972; Conte and de
K
Boor, 1980). On top of this is the variability in microorganism
In this latter case, for Salmonella in chicken broth (z = 5.9), the reductions which would render this approach unusable.
minimum time for sampling at K = 5 °C/min is 1.2 min. For Salmo-
nella in peanut butter (z = 56) and K = 5 °C/min, the minimum time
4. Conclusions
is 11 min. As for DT ref , Tables 4 and 5 show high accuracy and pre-
cision in general.
By combining the evaluation of DT ref and z, the number of exper-
One last detail concerning the isothermal method is tempera-
iments is cut by 75% from the traditional method of obtaining
ture lag. To impose a linear rate of increase on T, Text must also
these parameters wherein 4 data points are used to determine
change in a linear fashion. However, heat transfer resistance due
DT ref and three additional evaluations of DT ref are required to mea-
to the thermal properties of the heating medium and the thermal
sure z. Even greater savings are realized when more data points
death time device being used, causes the actual temperature to
are desired.
lag behind the measured Text. Therefore Eq. (10), which assumes
The mathematical development of three potential methods for
that T(t) is the temperature experienced by the bacterial suspen-
simultaneous determination of DT ref and z was presented and ana-
sion, needs to be modified when a lag exists. Since the lag will be
lyzed. The isothermal method, the first method discussed, evolved
constant, Text = T + Tlag. Substituting this into Eq. (10), and rearrang-
from the traditional method. Although it retained the experimental
ing gives:
! simplicity of the traditional method, the additional mathematical
T ext  T ref z T lag manipulation required to extract z was formidable, and was found
log R ¼ þ log  ð18Þ to be very sensitive to the normal variations expected in experi-
z KDT ref ln 10 z
mental data. The second method, the isochronal method, retained
From this equation it can be seen that the lag does not affect the the mathematical simplicity of the traditional method but was
value of z, but does affect the intercept, and therefore the value of experimentally more demanding. The last method, the non-iso-
DT ref . Including Tlag therefore allows the correct value of DT ref to be thermal method, retained both the mathematical and experimen-
calculated. It is important to note here that such a correction is only tal simplicity of the traditional method, while still yielding
8 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9

simultaneous values of DT ref and z. The method was also shown to A.3. Derivation of Eq. (15)
be robust with respect to expected variations in experimental data,
though with a minor adjustment for high z values. Starting with Eq. (A11), and taking the log of both sides, the fol-
lowing equation is obtained:
Appendix A. Derivations and Visual Basic Macros
z
log R ¼ log þ log½10ðTðtÞT ref Þ=z ð1  10ðTðtÞT 0 Þ=z Þ ðA12Þ
A.1. Derivation of Eq. (4) KDT ref ln 10

where the 10ðTðtÞT ref Þ=z term is seen factored out from within the
Starting with Eq. (2) and substituting in Eq. (3) gives, after some
parentheses. Separating the second term on the right hand side
rearrangement:
results in:
Z
10ðT ext T ref Þ=z t
f=s = ln 10
R¼ 10ae df ðA1Þ z TðtÞ  T ref
DT ref 0 log R ¼ log þ þ log½ð1
KDT ref ln 10 z
where
 10ðTðtÞT 0 Þ=z Þ ðA13Þ
T  T0
a ¼  lnð10Þ ext ðA2Þ When data is taken, even at large values of z, the 10 term is ðTðtÞT 0 Þ=z
z
on the order of 0.1. Substituting in the approximation
A change of variables is made using:
ln(1  x)  x, rewritten as log(1  x)  x/ln 10, yields Eq. (15)
u ¼ et=s ðA3Þ
A.4. Derivation of Eqs. (16) and (19)
to obtain:
Z uðtÞ Z uðtÞ Starting with Eq. (15) and taking the anti-log10 gives:
s 1 au= ln 10 s 1 au
R¼ 10 du ¼  e du ðA4Þ !
DT ref uð0Þ u DT ref uð0Þ u z Kt=z
R¼ 10ðTðtÞT ref Þ=z 1010 = ln 10 ðA14Þ
In Eq. (A4), use was made of the identity 10x = exln10. The solution to KDT ref ln 10
this integral is: Kt=z
The correction term, 1010 = ln 10 , should be near 1. Arbitrarily
!uðtÞ
s X
1
ðauÞi choosing a value of 1% or less as an acceptable error means that:
R¼ ln u þ ðA5Þ
DT ref i¼1
i  i! 1010
Kt=z
= ln 10
> 0:99 ðA15Þ
uð0Þ

Substituting back in Eqs. (A3) and (A2), then taking the limits, Solving this inequality for Kt=z, gives:
gives Eq. (4).
Kt
>2 ðA16Þ
A.2. Derivation of Eq. (9) z
Rearranging this gives:
When Eq. (8) is substituted into Eq. (2) and rearranged, the fol-
z
lowing equation results: t>2 ðA17Þ
Z K
ðT 0 T ref Þ=z t
10
R¼ 10K s=z ds ðA6Þ Thus, to keep the error term small, sampling times should con-
DT ref 0 form to Eq. (A17). When lag is important, Kt is replaced by Kt  Tlag.
The integrand in Eq. (A6) can be rewritten as:
A.5. Derivation of Eq. (21)
Z
10ðT 0 T ref Þ=z t
R¼ eðln 10ÞK s=z ds ðA7Þ Starting with the definition of z (Geankoplis, 2003):
DT ref 0
T2  T1
The integration is then straightforward: z¼ ðA18Þ
log DT 2  log DT 1
10ðT 0 T ref Þ=z ðln 10ÞK s=z t Substituting Text for T2 and letting T1 represent the actual suspen-
R¼ e ðA8Þ
DT ref Kz ln 10 0
sion temperature gives:

Switching back to base 10 and plugging in the limits: T ext  T 1

z¼ ðA19Þ
ðT 0 T ref Þ=z
log DT ext  log DT 1
10
R¼ K
ð10Kt=z  1Þ ðA9Þ Tlag is defined as the difference between Text and T1, so Eq. (A19)
DT ref z
ln 10
becomes:
Rearranging this equation yields:
T lag
z z¼ ðA20Þ
R¼ ð10ðKtþT 0 T ref Þ=z  10ðT 0 T ref Þ=z Þ ðA10Þ log DT ext  log DT
KDT ref ln 10
Rearranging this equation gives the desired result:
With Eq. (8), Eq. (A10) becomes:
DT ext
z T lag ¼ z log ðA21Þ
R¼ ð10ðTðtÞT ref Þ=z  10ðT 0 T ref Þ=z Þ ðA11Þ DT
KDT ref ln 10
 G.J. Fleischman / Journal of Food Engineering 155 (2015) 1–9 9

A.6. Visual Basic macro for the summation of the infinite series in Eqs. ‘
(4) and (6) Range(‘‘J11’’).Select ‘start with the intercept
difference in the
‘first row of experiments
Function sumn1(alpha, t, tau) For i = 1 To 100 ‘invoke GoalSeek 100 times
‘
‘Sums a series for calculating the log N0/N knowing
z and DT. ‘ Goal seek uses the present active cell (J11 to
start with)
‘Both t and tau are included to get the initial
curvature ‘
ActiveCell.GoalSeek
acc = 0.00001
sumn1 = 0 Goal:=ActiveCell.Offset(0, -1).Value,
n=1 _ChangingCell:=ActiveCell.Offset(0, -3)
factn = 1 ActiveCell.Offset(1, 0).Select
sumn1p1 = sumn1 + ((alpha ^ n) ⁄ (Exp(-n ⁄ t / tau) - Next
1)) / _ End Sub
(n ⁄ factn)
term = 1
While Abs(term) > acc
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