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International Journal of

Food Microbiology 30 (1996) 55-70


Application of predictive microbiology to estimate
the number of Bacillus cereus in pasteurised milk
at the point of consumption
M.H. Zwietering,*, J.C. de Wit, S. Notermansb
Department of Food Science, Wageningen Agricultural University, PO Box 8129, 6700 EV,
Wageningen, The Netherlands
bNational I nstitute of Public Health and the Environment, PO Box 1, 3720 BA, Bilthoven,
The Netherlands
Abstract
A procedure is presented to quantitatively estimate the growth of a particular organism in
a food product during chilled storage using predictive microbiology. This results in a
quantification of the contribution of every individual process step to the total number of
organisms, which may be a useful tool to support decisions on existing process lines as well
as in process and product design. It is demonstrated that predictive microbiology will only
estimate to within orders of magnitude of bacterial growth. This helps to pinpoint the most
important aspects of a line. The calculations can be helpful to set critical limits and to detect
hazards by performing what if analyses. The procedure is explained for the growth of
Bacillus cereus in milk. It is indicated, that with the current information, the effect of
time/temperature can be estimated. However, to make an accurate exposure analysis, more
information will be needed.
Keywords: Predictive microbiology; Milk; Bacillus; HACCP; Critical limits
* Corresponding author. Agricultural University, P.O. Box 8129 6700 EV, Wageningen, The Nether-
lands. Phone: + 31 317 484204, fax: + 31 317 482237. e-mail: Marcel.Zwietering@Algemeen@PK.
WAU.NL
0168-1605/96/$15.00 0 1996 Elsevier Science B.V. Ail rights reserved
PII SO168-1605(96)00991-9
56 M.H. Zwietering et al. / ht. J. Food Microbiology 30 (1996) 55 70
Due to the temperature applied during pasteurisation in milk the risks of
survival of vegetative pathogens and spoilage organisms are decreased consider-
ably. This is not the case for Bacillus cereus. The level of contamination with
this organism may be controlled by a low initial spore count, cooling after
pasteurisation, and limiting storage time. Both storage time and storage tempera-
ture are largely under control of the consumer. Therefore, the consumer is
informed about storage temperature and expiry date, which is presented on the
packages. From a risk analysis point of view, there is now interest to be in-
formed about, (i) the effect of time/temperature combination to control the level
of contamination and (ii) the consumer exposure upon consumption of pas-
teurised milk. There are several approaches to gather the necessary information,
including surveillance testing, storage tests and the application of predictive mi-
crobiology.
Predictive microbiology is a rapidly developing discipline, aiming at quantita-
tive estimation of microbial growth in foods using mathematical modelling. The
results of these models should not be relied on completely. They should be
regarded as tools to support decisions. They may be helpful to improve experi-
mental design and aid in the interpretation of experimental results. Moreover,
predictive microbiology can be helpful to decide which tests to perform and not
to put effort in things that are of little importance. Tools like microbial chal-
lenge tests, storage tests and surveillance tests may be just as useful. Together
these methods may provide better understanding and reliability of the predicted
results.
Predictive microbiology enables quantitative prediction based on the sequence
of events in the whole history of the product, and can be used to determine
what may happen in many different scenarios. However, predictions are based
on models and parameters which might not be correct in certain cases. Challenge
testing is carried out in those cases where the pathogenic organism is expected to
be present but only in low frequencies, indicating possible problems that may
occur. However, when an organism is able to grow in a certain product, it does
not always have to be a problem, since it may never be present in reality.
Storage tests give the actual growth in the product under study. However, rarely
found organisms might be overseen, and the actual storage conditions vary from
product to product, never exactly equalling the conditions of the storage test.
Surveillance tests give information on the actual numbers in the product at the
moment of consumption. It is however difficult to find out how these numbers
are reached and how they may be influenced. These methods are all giving
relevant answers, and the combination of these methods answers most of the
questions, i.e. the actual numbers and how they are reached, the effects of
various changes, etc. This information may be used as a basis for risk manage-
ment. In this paper predictive microbiology is applied to estimate the occurrence
of Bacillus cereus in milk and results are evaluated.
M.H. Zwietering et al. / Int. J. Food Microbiology 30 (1996) 55-70 51
2. Theory
2.1. Bacterial growth
For growth first order kinetics are assumed, possibly after a lag phase:
r,,
= ,uN (# .g-.h-) t>R
(1)
with Y, the organism production rate (growth in numbers per g per h), ,U the specific
growth rate (h _ ), N the concentration of organisms ( # /g), /i the lag time (h) and
# meaning number. In batch systems (which most foods are), the growth equals
the increase in numbers, resulting in:
(3)
N = N,exp[p(t - A)]
(5)
For prediction purposes this simple exponential model is chosen, since the accuracy
of the prediction will not justify the use of more sophisticated models (e.g.
sigmoidal).
2.2. Growth rate
The effect of temperature on growth rate is assumed to follow the square root
relation (Ratkowsky et al., 1982):
& = b(T- Twin) (6)
The b value is dependant on (among others) the pH and a, of the product and b
can be calculated on basis of these variables with the equation (McMeekin et al.,
1993; Wijtzes et al., 1995):
P = c(a),, - a
w,min)(PH - PHmi*KPHnux - PHMT - Tmin)2 (7)
For the parameters safe margins must be chosen, since for instance T,, is not the
lowest temperature in literature where growth is reported, it is the extrapolated
temperature at which no growth will occur. Since the growth rates around that
temperature are much too small to measure, the actual Tmj, will always be about
one degree lower than the minimal reported growth temperature (see Appendix 1).
The advantage of using this approach is that for every variable determining growth
rate (hurdle), the relative effect can be calculated, by separating the effects of the
various variables in Eq. (7), using the relative growth rate:
58
with:
M.H. Zwietering et al. / ht. J . Food Microbiology 30 (1996) 55-70
dT) = (;y-T;:i?)
(PH - PfL36Hmax - PW
y(pH) = cPf&, - ~fLi&fL, -P&J =
W - pHti,)(2 .P&, -P&C, - PW
(PHqz - PHmiJ 2
a, - %,h
Y@w) = l _ a
w,min
(10)
(11)
In a chilled food y(T) will usually be small, in an acid sauce this is true for y(pH),
and in bread for ~(a,). Knowing which variables may be used to influence bacterial
growth is useful of course, but for optimum use of this notion, avoiding putting
unnecessary efforts, the effects should be quantified. It should be noted that in these
equations there are no interactive effects of the various variables (T,;, is no
function of pH, pHti is no function of T, etc.), and the combined effect is assumed
multiplicative. So if y(T) = 0.1 and y(pH) = 0.1, the overall y = 0.01. Thus, 10%
of the maximum specific growth rate due to the temperature and 10% due to the
effect of the pH gives together 1% of the maximum specific growth rate. The fact
that very low growth rates cannot be easily measured, might even result in an
apparent dependence of the various variables (see Appendix 1).
For a food product with a given pH and a, the only adjustable variable is the
temperature. To describe the effect of temperature for this food product, the b value
of Eq. (6) may be estimated as follows:
(13)
This procedure, which is indicated as the gamma concept, needs only six
parameters (Tmi, Topt, PHmin, pHopt, aw,tin,
and popt) which are all easy to interpret,
and may be looked up in literature. These parameters can be easily adjusted if new
more tolerant strains emerge.
The (weighted) derivative of the gammas to the variable might also be of interest.
This gives the sensitivity of the growth rate to the variable, indicating how much
the growth rate may be changed with that variable (sensitivity) (see Appendix 2).
2.3. Predictions using the Pathogen Mode&g Program and Food Micromodel
Using the Pathogen Modeling Program (USDA, 1994) and Food MicroModel
version (Food MicroModel Ltd. Leatherhead Surrey UK), the growth of B. cereus
M.H. Zwietering et al. /ht. J. Food Microbiology 30 (1996) 55-70 59
can be predicted at various temperatures. For this the temperature, pH and
water activity (or salt concentration) must be entered. For the Pathogen Model-
ing Program a salt concentration of 1.3% (a, = 0.993) is entered and for Food
MicroModel a salt concentration of 1.2% (a, = 0.993) is entered, to result in
the same water activity.
2.4. Data based on storage tests
Results from storage tests obtained in the past (Dufeu and Leesment, 1974;
Griffiths and Phillips, 1990) and recently by us have been applied to compare
the various predictions. Own results were obtained in two different labora-
tories and two different experimental procedures (see Materials and methods,
Section 3).
3. Materials and methods
3.1. Lab 1
Semi-skimmed pasteurized milk (Campina Melkunie Woerden) was used. At
time zero the filling of the cartons was used. At four different moments a series
of cartons was taken from a milk processing line. Two series (one of 10 cartons
and one of 15) were stored at 7C and two at 10C (15 bottles and 5 car-
tons). After taking the cartons or bottles they were transported to the laboratory
at T < 7C. There they were placed at the desired incubation temperature
and sampling started next day. Samples were taken and the appropriate dilu-
tion was made. For counting the number of Bacilfus cereus MYP (mannitol
egg yolk polymyxin agar, prepared from Cereus selective agar base (Merck)
with egg yolk emulsion SR047C (Oxoid) and polymyxin-B-sulphate (B. cereus
selective supplement SR099E)) was used. The plates were incubated for 1 day at
30C.
3.2. Lab 2
Semi-skimmed pasteurized milk was used originating from different factories.
Immediately after filling, the cartons were transported to the laboratory at refrig-
erating conditions ( < 7C). In the laboratory 100 ml portions of each carton
was transferred to sterile 200 ml Erlenmeyer flasks and incubated at 6, 8, 10,
and 12C respectively. Sampling started next day and B. cereus were enumerated
as described above. Suspected colonies were confirmed by the following biochem-
ical reactions: gelatine ( + ), Voges Proskaner ( + ), nitrate reduction ( + ) and
glucose fermentation ( + ).
60 M.H. Zwietering et al. 1 ht. J. Food Microbiology 30 (1996) ST-70
4. Results
4.1. Predictions using the gamma concept
For B. cereus (T,i, = OC, T,,, = 37, pH,i = 4.9, pH,,, = 6.5, aw,,,in =
0.95, &pt
= 2 h-) in milk (T = 7C pH = 6.6, and a, = 0.993), the gamma
factors can be calculated with Eqs. (9)-(11) as: y(T) = 0.036, y(pH) = 0.996,
~(a,) = 0.86 (Table 1). The total y value is 0.031, so the growth rate is estimated
as 0.061. This gives a quantification of the hurdle concept. We can see from these
numbers that pH and a, give almost no reduction of growth rate (0.4% and 14%
respectively), and that the main reduction is achieved with temperature (96.4%).
For the example of Bacillus in milk the b-value may be estimated from Eq. (13) as:
b = 0.996.0.86- = /-T 0.0354
leading to an estimate for the growth rate:
J1 = O.O354(T- T,i,) = 0.0354. T
(14)
(15)
4.2. Comparison of the predictions
The results of the gamma concept, Food MicroModel (FMM), Pathogen Model-
ing Program (PMP-model), and literature data are presented in Table 2 and Fig. 1.
It should be noted that for any of these programs it only takes about 15 min to
produce results. Collecting and interpreting literature values takes about 1 day (8
h). Comparison of the growth rate predictions in Table 2 and Fig. 1 shows that the
various models and literature data agree very well. The PMP model shows too
small growth rates at low temperatures. However, it is clearly stated in the
program, that the parameters of the model for B. cereus are determined for a
mesophilic strain, and might not apply for psychrophilic strains. Considering this,
the predictions are not bad at all. The agreement between the various models and
the literature data shows that the concept of predictive microbiology can give
appropriate predictions.
Table 1
Data to calculate gamma for Bucihs in milk
Min
Opt
Actual y
T (C) 0 31 I (7 - 0)2/(37 - 0)2 = 0.036
PH 4.9 6.5 6.6 (6.6 - 4.9).(8.1 - 6.1)/(6.5 - 4.9) = 0.996
a, 0.95 1.00 0.993 (0.993 - 0.95)/0.05 = 0.86
p 0-l) 2.0
2 Total 0.031
Ratkowsky parameter b is equal to 0.0354
M.H. Zwietering et al. / ht. J . Food Microbiology 30 (1996) 55-70 61
Table 2
Growth rate (hh) predictions at various temperatures
T CC)
Dufeu and Leesment (1974) Griffiths and Phillips (1990) Gamma FMM PMP
3
6
8
10
17
25
30
38
0.0128
0.0555 0.0301, 0.0365
0.0462, 0.0578
0.0762
0.173, 0.231
0.289
0.408
0.533
0.147
0.0112 - -
0.0451 0.0524
0.0801 0.0794 0.0398
0.125 0.118 0.0686
0.361 0.415 0.325
0.782 1.34 1.02
1.13 2.42 1.53
1.807 - 1.89
Food MicroModel (Food MicroModel Ltd. Leatherhead Surrey U.K.).
bPathogen Modeling Program (USDA, 1994).
Program predictions are multiplied by 2.303[ln(lO)] to convert to specific growth rate (h- ).
4.3. Experimental ver$cation
The observed agreement between literature values and the various model calcula-
tions gives much confidence, however, to increase confidence, extra experiments
may be performed to determine the growth rate of Bacillus in milk. To perform
these experiments takes a lot of time. For testing 20 packages of milk applying four
incubation temperatures and a sampling frequency of every l-2 days during a
period of about lo-20 days, about 400 h are needed. The estimated growth rates
from those experiments where the distribution of data was well over all phases of
the growth curve (lag, exponential, and asymptotic phase), are plotted in Fig. 2 and
compared with the prediction of the gamma concept (Eq. (15)). In this graph it can
again be seen that these predictions agree very well with the experimental data.
4.4. Use of models to predict the number of bacilli in milk and comparison with
storage test results
Now that we have a model to predict the growth rate as function of temperature
we can make predictions of the growth in various cases. For the prediction of the
growth of Bucillus in milk we assume the absence of a lag phase. To estimate the
increase in numbers of organisms we need the following data: N,, b, Tminr
temperatures and residence times. The Ratkowsky parameters used, are those
predicted by the gamma concept (b = 0.0354, T,, = OC). We assume the initial
number of Bacillus present per bottle of 1 1 is 10 (N, = O.Ol/ml) (note that this
assumption is necessary, however, it can easily be checked what happens if there are
for instance 1 per ml).
To validate this procedure the experimentally determined number of organisms,
and the predictions at 6, 7, 8, 10 and 12C are given in Figs. 3-7. These figures
show that there is a large variation between various cartons of milk, as well as
62 M.H. Zwietering et al. / Int. J. Food Microbiology 30 (1996) 55-70
between various batches. However, the predicted numbers agree quite well, and
predict the correct order of magnitude for the increase of numbers in time. The
large variation between the different cartons shows that models need not be very
accurate for prediction (they can never be better than reality, and reality has large
variation in this case). The results from the two different laboratories show the
same behaviour.
4.5. Estimation of human exposure to B. cereus.
The number of organisms in various phases, of duration ti with temperature pi,
can be easily calculated with:
In(N) = ln(N,) + Cpiti = WV,) + Cb(T, - Ld2ti
(16)
i i
This equation can be converted to the logarithm with. base 10 yielding:
log(N) = log(N,) +
&zb2CTi - Tmin)2ti
I
(17)
1
/
/ ,/*
/
/ *
,/
0.8
=
c
F 0.8
$
c
3 0.4
$
0.2
0
0 5 10 15 20 25
temperature (OC)
Fig. I. Comparison of the predicted growth rates of various models and literature data. n Griffiths and
Phillips (1990); + Dufeu and Leesment (1974); ~- Gamma model; - - - Food MicroModel (Food
MicroModel Ltd. Leatherhead Surrey U.K.); PMP model (USDA, 1994).
M.H. Zwietering et al. / ht. J . Food Microbiology 30 (1996) 55-70 63
0 5 10 15
temperature (C)
20 25
Fig. 2. Comparison of the predicted growth rate of the Gamma model and newly performed experi-
ments. A experiment; -- Gamma concept.
Using the above given parameters and converting the time from hours to days, this
equation becomes:
log(N) = - 2 +
(0.0;354);.24TT?ri
(18)
Alog = O.O130CTf&
(19)
Lets assume the following scenario: company storage (2 days, 4C), retail (2 days,
7C) consumer storage (5 days, 7C). We can predict now the number of organisms
in all the stages (Table 3). These calculations can be very easily set up in a
spreadsheet program and it is clear that all kind of scenarios can be calculated this
way, showing the relative importance of the various phases, and pinpointing the
critical stages. Limits may be set for the various times and temperatures in a
quantitative way. This procedure can be extremely useful for a what if analysis.
For instance the remaining shelf life for various storage conditions (T,, t,) can be
calculated using Eq. (19), and the conditions in the company and at retail as given
in Table 3:
log(N) = log(N,) + 0.0130(16.2 + 49.2 + T,2t,)
If we set as limit N = lo5 this results in:
(20)
64 M.H. Zwietering et al. / ht. J . Food Microbiology 30 (1996) 55-70
5 - lo
0.0130
- 32-98 = Tzt,
(21)
Some results for various values of N, and T, are given in Table 4. It is clearly
demonstrated that the storage time depends both on the initial contamination and
the storage temperature. From this analysis it can be concluded that the relative
effect of each phase is equal to (T, - T~n)*ti (in this case T,i, was equal to zero
resulting in T2ti). So by just using the minimum growth temperature of the relevant
microorganism, and the temperature and the residence time, the relative effect of
the various phases can be determined.
From the foregoing it has become clear that if the history (initial B. cereus count,
storage time and storage temperature) of the pasteurised milk is known, the number
of organisms can be estimated. However, this only applies for the optimum growth
conditions. As a consequence a worst case result is obtained. Another aspect is that
there are indications that the initial count in milk is season dependant (own
observations) which thwarts an exact calculation. Furthermore, the storage temper-
ature and time of the milk for an individual consumer are difficult to estimate.
These data may be obtained by sentinel studies. These studies will show that both
the storage temperature and time will show a statistical distribution. The fact that
these distributions may be dependent, again complicates an exact estimation of
human exposure.
6-
-4
f. I ,
0 200 400
time (h)
600 800
Fig. 3. Comparison of the predicted number of organisms and the measured number at 6C. Various
symbols give various cartons (lab 2); __- Model prediction.
M.H. Zwietering et al. / ht. J. Food Microbiology 30 (1996) 55-70 65
6
0 400 600
time (h)
Fig. 4. Comparison of the predicted number of organisms and the measured number at 7C. n Series
1 (lab 1); + Series 2 (lab 1); __- Model prediction.
5. Conclusions
Predictive models may give a useful estimation of the growth of spoilage and
pathogenic organisms in foods. The predictions must, however, always be critically
evaluated, and not be relied on exclusively. Various modeis, literature data and
storage tests are in very good agreement. The models can be used to calculate the
effect of various storage scenarios. The use of these models is easy, costs very little
time and can give much insight. Comparing model predictions with literature data
may add confidence to the most relevant assumptions (the difference in time of
making model predictions and searching literature storage tests is about a factor
30).Finally, well designed storage, surveillance or challenge testing can be used to
increase the confidence to a desired level. Since this takes about a factor 1000 more
in time, these tests should be well designed and must not be done if not necessary.
The storage tests appeared to show very large variation between various cartons.
So, there is no need to predict the growth by any sigmoidal curve. In the prediction
of the growth only an order of magnitude prediction can be obtained. It is then not
relevant to describe exactly the curves in the growth response. The simple exponen-
tial growth model is sufficient. For data treatment of individual growth curves
sigmoidal models are useful, however. One individual growth curve shows a smooth
transition for lag to exponential and exponential to stationary phase. Therefore,
models to describe one set of growth data need to show sigmoidal behaviour.
66 M.H. Zwietering et al. /ht. J . Food Microbiology 30 (1996) 55-70
Of course many other important factors can be included in this procedure. For
instance the experimental determination and possible implementation of the lag
time can be a next step. Also the experimental determination of the (statistical
distribution of the) starting level will give the predictions a better basis. The
variation in temperatures and in residence times can be implemented and the effect
of these can be determined for instance by Monte-Carlo simulation. Maybe such
more detailed studies may explain the variation in numbers as found in this study.
If the cause of the variation is known, maybe it can give directions to diminish it.
This is only possible if the cause of this variation lies in controllable circumstances.
However, it might be that the variation is caused by biological variation (variability
of the state of the organism after the heat treatment, variation in sporulation).
Biological variation is difficult to influence, and we have to live with that. In that
case, simple calculations as given in this paper may still provide an effective
quantification of the number of Bacillus during storage. This then can be used to set
critical limits, determine hazards, and to design relevant experiments.
Together with storage tests, surveillance tests, sentinel studies, and dose response
relations, predictive microbiology may be employed to get an estimate of human
risk from pathogenic microorganisms. All these methods must deal with the
problems of inaccuracies, and statistical variability. Yet, in certain cases, decisions
must be made. These decisions can be based on experience. To lend quantitative
6
0
-2
time(h)
406 600
Fig. 5. Comparison of the predicted number of organisms and the measured number at 8C. Various
symbols give various cartons (lab 2); ~ Model prediction.
M.H. Zwietering et al. / ht. J. Food Microbiology 30 (1996) 55-70
67
10
8
6
z
4
s
-0
2
0
-2
-4
/
L
l l l
:
l
1
0 200
time (h)
400 600
Fig. 6. Comparison of the predicted number of organisms and the measured number at 10C. n Series
1 (lab I); + Series 2 (lab 1); A Series 3 (lab 2); -- Model prediction.
6
4 6 8 10
time (h)
Fig. 7. Comparison of the predicted number of organisms and the measured number at 12C. Various
symbols give various cartons (lab 2); __ Model prediction.
68 M.H. Zwietering et al. 1 ht. J . Food Microbiology 30 (1996) 55- 70
Table 3
Calchlation of the number of organisms, given a certain scenario
Phase
Initial
Company
Retail
Consumer
T(C)
Time (days)
4 2
7 2
7 5
T2t
d lW(W h&V
-2
32 0.417 - 1.6
98 I .278 -0.3
245 3.196 2.9
Table 4
Results of a what if analysis. Storage times (in days) giving a final level of log(N) = 5 (the conditions
in the factory and retail are the same as in Table 3)
Initial number (NJ Consumer storage temperature (C)
6 7 8 9 10 11 12
0.001 13.4 9.9 7.6 6.0 4.8 4.0 3.4
0.01 11.3 8.3 6.4 5.0 4.1 3.4 2.8
0.1 9.2 6.7 5.2 4.1 3.3 2.7 2.3
I 7.0 5.2 4.0 3.1 2.5 2.1 1.8
10 4.9 3.6 2.8 2.2 1.8 1.5 1.2
support to these decisions, models as presented in this paper, can be used. For a
start, the decision may be on the safe side (worst case). To move away from this
safe estimate, ever more specific information must be gathered.
Acknowledgements
The authors would like to thank Dr. Buchanan- for his free donation of the
Pathogen Modeling Program and Food MicroModel for the permission to use
predictions. We thank I. Dijkstra and J. Dufrenne for the experimental work. Part
of the experimental work was carried out at Campina Melkunie, and we would like
to thank-J. Dijkers for making this possible.
Appendix A
It should be noted that for the parameters safe margins must be built in, since for
instance Tt i , is not the lowest temperature in literature where growth is reported,
it is the extrapolated temperature at which no growth will occur. Since the growth
rates around that temperature are much too small to measure, the actual Tmi , will
always be about one degree lower than the minimum reported growth temperature.
If we assume an experiment that lasts six tionths (without a lag phase) and we
M.H. Zwietering et al. /ht. J.
assume that the detection of growth is a
rate is
In $ = ln(2) = ,ut
0 0
ln(2)
__ = 1.58~10-4h-
= 182.24
& = &%%= = 0.0126 =
Food Microbiology 30 (1996) 55- 70 69
factor two, the minimum detectable growth
(22)
(23)
b(T-T,,,,,) = b.AT
(24)
With a b value of 0.0354 (Eq. (15)) this results in difference between Tmin and the
minimum detectable growth temperature of:
0.0126
AT=----=
0.0354
0.36
(If the experiment would only have been pursued for 20 days, the difference would
be 1.07 degrees).
Often at these low temperatures there will be a lag phase, which is of the order
of twice the inverse of the growth rate (between zero and 2/p)
ln(2) = ,~(t - /i) = p. t - p. ;1 x p. t - 2
(26)
,u .t = 2.69
(27)
2.69
___ = 6.13.10-4
= 183.24
(28)
4; = dm = 0.0248 = bAT
With a b value of 0.0354 for Bacillus (Eq. (15)) this results in:
(29)
0.0248
AT=-----=
0.0354
0.70
(If the experiment would only have been pursued for 20 days, the difference would
be 2.12 degrees),
At less fdvourable conditions (lower pH or a,) the b value decreases and the
difference between the minimum growth temperature and the detectable minimum
growth temperature becomes larger. This might result even in an apparent depen-
dency between the minimum growth temperature and for instance the a,. For
instance for a product of a, = 0.96 and optimum pH the b value becomes:
b=
J
(a+r,f?;mi,) =
J
(0.96 - 0.95). 2
(1 -0.95) (37-0)2 = 0.0171
(31)
AT = 0.0248
- = 1.45
0.0171
(32)
(If the experiment would only have been pursued for 20 days, the difference would
be 4.38 degrees).
70 M.H. Zwietering et al. / ht. J . Food Microbiology 30 (1996) 55-70
Appendix B
The derivative of the gammas to the variable can be calculated as:
Y = Y(T)YW)Y(G)
dy MT)
dT = ~YWJ)YW =
WT) Y
dT Y(T)
(33)
Therefore, the specific (relative) change can be defined:
1 dy 1 &G)
-=
ydT Y(T) dT
(34)
The derivatives are:
MT) W- Tmi,) &Wf) dy(d
1
-= -=
XP& - PH) _
dT (T,,z - Twin)* dpH WC,,, -P%,)* da, l - %,min
(35)
The specific (relative) changes are:
1 dy 1 MT)
2
ydT= y(T) dT =(T- Tmin)
1 @OH) 2(PH,,, - PH)
Y (pff) dpH = @H - PHmd(2PHo~t - PHmin - PH)
1 Ma,)
1
Y(G) da, = a, - G,min
(36)
The derivatives to the three variables can be weighted, since it is easy to change the
temperature by one unit, however, it is impossible to change the a, one unit. The
weights could be 1 for temperature, 0.2 for pH and 0.002 for a,. These weights are
determined from a range in temperature between 0 and 25 (weight l), pH between
4 and 9 (weight 5/25), and water activity between 0.95 and 1 (weight 0.05/25). This
means that 1 degree, 0.2 pH unit and 0.002 a, unit are equally desired or possible.
These weighted specific changes can be used to determine the possibilities to change
growth rate or shelf life with a certain variable.
References
Dufeu, J.N. and Leesment, H. (1974) Growth and resistance characteristics of some psychrotrophic
sporeformers isolated from raw milk. XIX International Dairy Congress, India, Vol. 1, pp. 535-536.
Griffiths, M.W. and Phillips, J.D. (1990) Incidence, source and some properties of psychrotrophic
Bacillus spp found in raw and pasteurized milk. J. Sot. Dairy Technol. 43, 62-66.
McMeekin, T.A., Olley, J.N., Ross, T. and Ratkowsky, D.A. (1993) Predictive Microbiology. Wiley,
Chichester, UK.
Ratkowsky, D.A., Olley, J., McMeekin, T.A. and Ball, A. (1982) Relationship between temperature and
growth rate of bacterial cultures. J. Bacterial. 149, 1-5.
USDA, (1994) Pathogen Modeling Program version 4.0. Microbial Food Safety Research Unit. USDA
ARS Eastern Regional Research Centre. Philadelphia, USA.
Wijtzes, T., de Wit, J.C., Huis int Veld, J.H.J., vant Riet, K. and Zwietering, M.H. (1995) Modelling
bacterial growth of Lactobacillus curvatus as a function of acidity and temperature. Appl. Environ.
Microbial. 61, 2533-2539.