LWT - Food Science and Technology 68 (2016) 532e540

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LWT - Food Science and Technology

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

Development of a freshness-assessment model for a real-time online

monitoring system of packaged commercial milk in distribution
Byeong-Sam Kim a, Minwoo Lee b, Ji-Young Kim a, b, Jae-Yoon Jung c, Junemo Koo b, *
a
Food Marketing/Distribution Research Group, Korea Food Research Institute, Baekhyeon-dong, Bundang-ku, Sungnam, Gyeonggi 463-746,
Republic of Korea
b
Department of Mechanical Engineering, Kyung Hee University, 1732, Deogyeong-daero, Giheung-ku, Yongin, Gyeonggi 446-701, Republic of Korea
c
Department of Industrial and Management Systems Engineering, Kyung Hee University, 1732, Deogyeong-daero, Giheung-ku, Yongin, Gyeonggi 446-701,
Republic of Korea

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

Article history: The freshness of packaged pasteurized milk under fluctuating temperature condition in distribution,
Received 13 July 2015 which was assessed by measuring the total microbial count in milk samples, was monitored by applying
Received in revised form realetime temperature logging technology, which included RFID (Radio Frequency Identification), WSN
15 November 2015
(Wireless Sensor Networks) and predictive microbiology. The freshness assessment model, which would
Accepted 21 December 2015
Available online 24 December 2015
be used in a real-time online quality monitoring system for packaged commercial milk, was developed
considering the impacts of large thermal capacity of packaged milk, wide biokinetic temperature range,
i.e., 5e30
C, and intermediate lags with temperature shifts as well as the choices of the primary and
Keywords:
Predictive modeling
secondary models such as the Gompertz, the Roberts and Baranyi, and the Hills and Wright models. The
Microbial growth justifiability of considering additional model parameters was statistically analyzed using Bayesian In-
Dairy formation Criterion (BIC) to select the best freshness assessment model. The freshness assessment model
Fluctuating temperature with the Hills and Wright model as the primary model and the two-zone Arrhenius model as the sec-
Shelf-life ondary model was selected as the best one to use for the real-time milk freshness monitoring system.
Spoilage © 2015 Elsevier Ltd. All rights reserved.
Safety

1. Introduction There is a higher probability that milk will experience a different

Milk freshness is determined by factors that are related to both
the production and the distribution processes. The initial quality of
packaged milk, including the freshness, is controlled by manufac-
turers. Once the milk is packaged into vessels, the intrinsic pa-
rameters that affect its freshness, such as pH, water activity,
nutrient availability, and initial microbial contents, are not subject
to further modification. Considering that the packaging protects the
milk from exposure to external air and humidity, the most impor-
tant factor affecting milk freshness is temperature. In circulation,
packaged milk is usually loaded onto delivery vehicles with a
temperature control system, and then the milk is displayed for sale
by sellers. Milk should be sold and consumed within a specified
shelfelife, which is usually estimated as the time required at a
constant temperature for the microbial concentration to reach a
critical value. However, food spoilage accelerates with temperature.
* Corresponding author.
E-mail address: jmkoo@khu.ac.kr (J. Koo).
temperature history during the distribution stage than during the
production stage, which might have a detrimental impact on
freshness. The effects of the temperature history on microbial
growth have been investigated by many researchers. Wells and
Singh (1988) developed a kinetic model to predict the changes in
tomato firmness based on a complete temperature history. Fu,
Taoukis, and Labuza (1991) and Labuza and Fu (1993) summa-
rized the application of predictive microbiology to determine and
predict the shelf-life of perishable food using the microbial growth
kinetics. The authors provided details regarding how to apply this
theory to fluctuating temperature conditions and a description of
the time-temperature indicator, which provides information on the
temperature history using a mechanical, chemical or enzymatic
system. Sørhaug and Stepaniak (1997) published a review article on
the impact of psychrotrophs and enzymes on the milk freshness.
Zygoura et al. (2004) investigated the effects of the packaging
material on milk freshness. Corradini, Ame zquita, Normand, and
Peleg (2006) reported that they could successfully generate mi-
crobial growth curves under complicated thermal histories
including regular and irregular temperature oscillations with the

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 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540 533

assumption that the organ's momentary non-isothermal growth for the milk stored under different temperature conditions, i.e., 0,
rate is the isothermal rate at the given temperature at a time that 10, 20, 30 and 40
C. The absolute correlation values of pH and
corresponds to its instantaneous population size. They also acidity with organoleptic test were over 0.8 for the temperature
demonstrated that as long as the empirical primary and secondary conditions over 10
C, whereas it was below 0.55 under 0
C con-
models capture the isothermal expressions of very different con- dition. The correlations between microbial concentration and
struction could be used interchangeably to predict the growth organoleptic test were 0.81 under the lowest temperature condi-
patterns under non-isothermal conditions. tion, and 0.99 under the highest temperature condition. The change
Although there have been many studies using predictive in the microbial concentration was selected as the best indicator for
microbiology to elucidate the effects of temperature on the mi- milk freshness not only because the correlations are better but
crobial growth of a selected bacterial strain in small samples, the because the correlations are better for lower temperature condi-
application of this approach on packaged milk under fluctuating tions under which milk is normally distributed.
temperature conditions remains limited. Alavi, Puri, and Mohtar
(2001) performed a computational heat transfer study to investi- 2.1. Development of microbial growth prediction model
gate the effect of the temperature distribution in a commercial
carton of pasteurized milk on the growth of Listeria monocytogenes. Microbial growth models are composed of the primary and
The authors used the Baranyi and Roberts model for the prediction. secondary models. The primary models mimic the sigmoidal nature
Subsequently, Xanthiakos, Simos, Angelidis, Nychas, and of microbial growth in time, and the secondary models represent
Koutsoumanis (2006) noted that models developed by other re- the microbial growth rate change with temperature in a functional
searchers had been developed under well-controlled laboratory form.
conditions and using microbiological media. Thus, it was not sur- For the primary model, Gompertz, Baranyi and Roberts, and Hills
prising that the models failed when applied to actual food samples. and Wright models were tested. The differential equation form of
They attributed this failure to the impact of factors such as the food the Gompertz model is shown in Eqs. (1) and (2):
composition and the microbial competition in real food, which
were disregarded in the previous experiments of other researchers. dy emmax
em 
max
¼ $exp ðl  tÞ þ 1 $yðtÞ (1)
Psomas, Nychas, Haroutounian, and Skandamis (2011) developed a dt A A
tertiary model that integrates primary and secondary models to
 
understand the microbial behavior in foods, which was based on NðtÞ
the Baranyi and Roberts model. Chen, Wei, and Chen (2011) yðtÞ ¼ ln (2)
N0
analyzed the source of the spoilage of ultra-high temperature
(UHT) pasteurized milk in cold storage using the modified Gom- where N, N0, Nmax, mmax, l, t and A represent the current microbial
pertz equation. concentration, the initial microbial concentration, the maximum
The real-time online freshness monitoring system of packaged microbial concentration at the stationary phase, the maximum
commercial milk in distribution under development is outlined as growth rate at a given temperature, the lag phase duration, the
following: RFID/WSN technology is combined with predictive current time, and the model parameter defined as ln(Nmax/N0),
microbiology and shelf-life prediction technologies to monitor the respectively.
quality of commercial milk from shipping to shelf. The milk tem- The differential equation form of the model (Baranyi and
perature is sampled once every 10 min by radio frequency tem- Roberts., 1994; Swinnen, Bernaerts, Dens, Geeraerd, & Van Impe,
perature sensor tags in the high-frequency band, i.e., 424e433 MHz 2004) is shown in Eqs. (3) and (4).
attached at the bottom of each milk vessel, and sampled temper-  
ature history data are transmitted by a CDMA and GPS communi- dN Q ðtÞ NðtÞ
¼ $mmax $ 1  $NðtÞ (3)
cation network to a web-server. A quick response (QR) code is dt 1 þ Q ðtÞ Nmax
attached on the surface of milk vessel to identify the milk. When a
consumer tries to scan the QR code on a milk vessel using his/her dQ
cell phone to check the freshness of the milk, it links to the web-site ¼ mmax $Q ðtÞ (4)
dt
where the milk freshness information is displayed based on the
prediction of the food freshness assessment model presented in where Q is a parameter representing the physiological state of the
this study considering the temperature history of the milk to pro- food.
vide consumers, providers and distributors with the real-time food Eqs. (5)e(7) show the set of equations of the Hills and Wright
freshness information and timely guideline for the proper con- model.
sumption and distribution of the products. Refer to Kim et al. (2011)
for details. dN
¼ kn ðTÞ$sðtÞ$NðtÞ (5)
In this study, the new findings are presented with a focus on dt
issues related to food freshness assessment model. These issues
include the following: temperature deviation at the milk center ds
¼ ðmmax ðTÞ  kn ðTÞ$sÞð1 þ sÞ (6)
from the set temperature due to the large thermal capacity of the dt
packaged milk, the impact of intermediate lag, and the develop-
ment of the milk freshness assessment model considering the two kn ðTÞ ¼ ðkn Þ0 expðEa =TÞ (7)
issues.
where kn is a parameter that is proportional to the rate of DNA
2. Material and methods synthesis, and s represents the excess biomass in a cell with its
initial value zero, i.e., s ¼ 0 at t ¼ 0 (Hills & Wright, 1994; Swinnen
Variations in microbial concentration, pH, and acidity with time et al., 2004). In general, kn in Eq. (6), together with the maximum
were tested under constant and fluctuating temperatures as growth rate mmax, governs the intermediate lag and varies according
possible indicators of milk freshness. The correlations between the to the environment, i.e., the temperature and/or temperature jump.
organoleptic test results and the three indicators were compared With a temperature shift, the maximum growth rate mmax in Eq. (6)
534 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540
changes and the term in the first parenthesis of the right-hand side
deviates from zero and changes the variable s until its change rate
becomes zero to consider the intermediate lag. The Hills and
Wright model could not depict the stationary phase due to the lack
of 1  N/Nmax term in it, but it could be used to monitor milk
freshness because the allowable level of microbial concentration in
milk is much lower than that in the stationary phase.
Arrhenius and square root models, which assume that the mi-
crobial growth rate increases either exponentially or in the manner
of a square function over a narrow temperature range were
compared as the secondary model (Ratkowsky, Lowry, McMeekin,
Stokes, & Chandler, 1983). For a short temperature range of inter-
est, assuming the frequency factor of Arrhenius model, A0 ¼ exp(A),
and Ea/R ¼ B, Arrhenius model could be represented as following:
   
Ea B
mmax ¼ A0 exp  ¼ exp A 
RTðKÞ TðKÞ
where A0 , Ea, R, T, and B stand for frequency factor, activation energy,
universal gas constant, temperature and a model parameter
respectively. Eq. (9) presents the Ratkowsky model, or the square
root model which is commonly used as the secondary model.
pffiffiffiffiffiffiffiffiffiffiffi
mmax ¼ bðT  Tmin Þ
Here, b is a constant, and Tmin represents the minimum tempera-
ture for microbial growth.
2.2. Milk sampling and microbiological analysis
The milk used in this study was commercially packaged
pasteurized milk (63
C for 30 min) that was obtained from a
commercial dairy on the day of production, and the milk was
transported to the laboratory under cold conditions within 24 h for
analysis. The main purpose of the current research is to develop an
online monitoring method of the temperature abuse for packaged
milk in real circulation using predictive microbiology. In summer,
the temperature in a cabinet of a delivery truck can rise easily
higher than 30
C, if the chiller goes out of order. The upper-
temperature limit, 30
C, was selected to consider the worst-case
scenario under the real circulation condition. The milk samples
were collected eight times from August 2010 to May 2012. After
transportation, each sample was stored at 5, 10, 15, 20 and 30
C and
submitted to microbiological analyses for the isothermal experi-
ments. The samples stored at 5, 10 and 15
C were analyzed every
24 h, and the sample stored at 20
C was analyzed every 6 h. The
samples stored at 30
C were analyzed every 2 h. Additional sam-
ples were exposed to fluctuating temperatures by incubation either
in a temperature-controlled chamber or in a refrigerator with a
programmable temperature history function. The number of rep-
licates was six for each sampling. Following incubation at a range of
temperatures fluctuating from 5 to 20
C, the samples were
analyzed every 2 h.
According to Duyvesteyn, Shimoni, and Labuza (2001), the shelf-
life of milk is traditionally estimated by both total and psychro-
trophic microbial counts. Walkling-Ribeiro, Rodríguez-Gonza lez,
Jayaram, and Griffiths (2011) also performed the shelf-life estima-
tion using aerobic microbial counts. In Korea, the shelf-life of
packaged milk is guided by Korea Consumer Agency (KCA) and
Animal, Plant, and Fisheries Quarantine and Inspection Agency
(QIA) to be estimated by the time when the total microbial count
reaches 2  104 CFU/mL (QIA, 2012). Therefore, the total microbial
count is selected to be a good index to monitor the freshness of
packaged milk complying with the government guidance.
To determine the total aerobic counts for each sample, 10 mL of
milk was aseptically transferred into a sterile stomacher bag, and
90 mL of a sterile 0.85% saline solution was added. The sample was
then homogenized in a stomacher (Bag Mixer 400, Interscience,
France) for 1 min at normal speed (8 strokes/sec), and aliquots were
plated out directly as 10-fold dilutions in a 0.85% saline solution.
Petri-film aerobic count plates (3M Microbiology Products, St. Paul,
MN) were inoculated, in duplicate. One mL of each dilution applied
in the center of the plate and between the two films. The inoculant
was covered with the upper film and expanded with a plastic
disperser over an area of 20 cm2. The plates were incubated at 35
C
for 48 h. All plate counts are expressed as the number of colony-
forming units per milliliter (CFU/mL).
2.3. Temperature logging
(8) Three different methods were used to log the milk temperature.
Wired temperature loggers (Model TR-5i; T&D Corporation, Japan)
of ±0.3
C accuracy and 0.1
C resolution were used to monitor the
temperature at the center of each milk package. The newly devel-
oped RFID temperature sensors use humidity and temperature
sensor IC (SHT21, Sensirion, Switzerland), which has 0.01
C reso-
lution and 0.3
C accuracy tolerance. RFID temperature loggers
were attached to the milk package surface, and the surrounding
(9)
temperature was monitored by either the wired temperature log-
gers or the temperature sensors installed in the refrigerator. The
thermodynamic model to relate food center temperature and the
new RFID temperature sensor is under development considering
the convection heat transfer between the surrounding and milk
package as well as the conduction heat transfer through the milk
package. In this study, the freshness assessment model has been
developed using only the temperature histories logged by the
wired temperature loggers.
2.4. Comparison between observations and model fittings/
predictions
The deviation between the fittings/predictions and the experi-
mental observations was estimated in the form of Af and Bf in Eqs.
(10) and (11), which are the accuracy and bias factors, respectively
(Ross, 1996).
 
2P ypredicted 3
log y
4 5
observed
n
Af ¼ 10 (10)
 
2P 3
log
ypredicted
yobserved
4 n
5
Bf ¼ 10 (11)
The bias factor Bf is used to show how well the model predicts
the measurement-mean variation for microbial growth, whereas
the accuracy factor Af represents the scattering of the data. The two
terms have a value of one when the fittings/predictions of the
model perfectly match the means of the experimental observations
and the experimental data have no scattering, respectively. The bias
factor could be either greater or smaller than unity depending on
whether the model overestimates or underestimates the observa-
tions. The accuracy factor is always greater than one by definition.
According to Pe rez-Rodríguez and Valero (2013), the acceptable
bias factor value for a predictive model could be 0.75e1.25. They
reported that the given criteria have been used in various validation
studies.
In this study, the accuracy and bias factors were estimated for
 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540 535

the data in the time range between the initial time to monitor and in Eq. (3), and the model was selected as a viable model to monitor
the time when the microbial growth reached the guideline mi- microbial growth in milk under both constant and fluctuating
crobial concentration for the shelf-life determination, 20,000 CFU/ temperature conditions.
mL.

3.2. Effects of the finite thermal capacity of packaged milk

2.5. Selection of the best food quality monitoring model

Previous experimental tests to determine the parameters in the

Since there are many choices for the primary and secondary
models like the Gompertz, the Baranyi and Roberts, and the Hills
and Wright models for the primary model, as well as the Arrhenius,
and the Ratkowsky models for the secondary models, for example,
it is possible to make many combinations between them. If the
numbers of model parameters and considered data for fitting are
the same, the best model set could be selected based on the coef-
ficient of determination (r2), the squared sum of residuals (SSR), Af,
and Bf. If not, we could not compare the models directly using them.
Therefore, there should be a criterion to select the best combination
of models to describe the microbial growth. Quinn and Keough
(2002) stated that a desired characteristic of the criterion is that
it could protect against overfitting, where the addition of extra
parameters may suggest a better fit even when the added param-
eters add very little to the explanatory power. They compared four
criteria to select the best model set including adjusted r2, Mallow's
Cp, Akaike Information Criterion (AIC), and Bayesian Information
Criterion (BIC), and they reported that BIC was the most reluctant
criterion to allow additional parameters. The definition of BIC is
shown in Eq. (12).
BIC ¼ n logðSSRÞ þ ðp þ 1Þlog n  n log n
where n and p represent the numbers of data used for fitting or
prediction and model parameters respectively. Given any two
estimated model sets, the model set with the lower BIC is better. If
the two models to compare use the same data sets, and the
numbers of model parameters are the same, the explanatory power
of the models can be compared directly using SSR. If the two
models have different numbers of model parameters, the second
term in Eq. (12) would impose a penalty for the addition of addi-
tional parameters. BIC will decrease only if the reduction of SSR
outweighs the penalty for the additional parameters. In this study,
BIC was used to compare between the tested model sets, and to
avoid overfitting in the model selection.
3. Results and discussion
primary model have primarily been carried out using small sample
volumes, typically less than 20 mL. A negligible amount of time was
required for the milk to reach the target test temperature, and the
constant temperature history assumption holds. For example,
Zwietering, de Wit, Cuppers, and Van’T Riet (1994) reported that it
took only 16 min for the 10 mL sample to reach the new thermal
equilibrium during the temperature shifts. However, for commer-
cially packaged milk in containers of 200 and 1800 mL, a finite
period of time was required for the deviation between the target
and food temperature to become negligible, and this factor should
be considered in determining the primary model parameters. Ex-
amples of the milk temperature history deviation from the set
constant temperature history are presented in Fig. 1. Although the
experiments were performed under the constant temperature
conditions, the milk temperature varied due to the thermal ca-
pacity of the milk in the container, and it approaches the target
temperature in a simple exponential manner following the New-
ton's law of cooling. If the temperature deviation shown in the
figure was not considered appropriately, the microbial growth
might have been underestimated. To handle this problem, the pa-
(12) rameters of the primary and secondary models were determined
simultaneously by using DEoptim and minpack.lm packages in the
open source statistics package, R (R Core Team, 2014) to minimize
SSR of the logarithmic microbial counts between the fittings and
the experimental observations. The DEoptim package uses the
differential evolution optimization algorithm, and minpack.lm
package is developed based on the LevenbergeMarquardt algo-
rithm. Fig. 2 depicts the flowchart to determine the three param-
eters included in the combination of the Baranyi and Roberts and
the Arrhenius models (BRA1). The boxes and arrows with dotted
lines in the figure represent the parts where DEoptim and min-
pack.lm packages are used to adjust the model parameters to
minimize the deviation between fittings and experimental obser-
vations. The temperature history was obtained from the tempera-
ture logger, and the logarithmic non-dimensional microbial

3.1. Selection of the microbial growth model

In this study, the Baranyi and Roberts, and the Hills and Wright
models were selected over the Gompertz model due to the rigor-
ousness of the modeling. From its definition y(t ¼ 0) is zero, and its
first derivative in time is zero as shown in Eq. (1), resulting in the
trivial solution, y(t) ¼ 0. This problem does not affect the fit of the
model for the isothermal condition because the integrated form of
the equation is used in that case. However, this problem does occur
under fluctuating temperature conditions, when the differential
form of the model should be used. To avoid this, a very small
number was assumed for y(0), e.g., 1020 or 10100. The milk
freshness histories were very different, even for a negligibly small
difference in the initial microbial concentration value, which did
not support the real phenomena. The Gompertz model was found
to be not applicable for the fluctuating temperature cases. A similar
issue was discussed by Garthright (1991). The Baranyi and Roberts
model was found to be a better fit for the fluctuating temperature
conditions. In contrast to the Gompertz model which has zero
slopes at the initial point, the Baranyi and Roberts has a finite slope Fig. 1. The deviation between the set and real temperature histories.
536 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540
Fig. 2. Flowchart to determine the model parameters.
number and history were determined by integrating Eqs. (3), (4)
and (8) using the second order Runge-Kutta method with the
assumed model parameters. The optimum set of model parameters
was determined from the iterative process to minimize the sum of
squared deviations of the logarithmic microbial counts between
the fittings and the experimental observations. This process is
called system identification, which uses statistical methods to build
mathematical models of dynamical systems from measured data.
The accuracy of models was compared between the models with
and without the consideration of temperature history. For the case
of neglecting the temperature history, it was assumed that the
temperature was maintained at the mean values of the temperature
history for each case throughout the test. The microbial count at the
stationary phase was measured to be 1.678  107 CFU/mL on
average with the standard deviation of 3.463  106 CFU/mL. In the
model fitting calculation, Nmax was fixed at the average value, while
the mean value of N0 measurements from each constant temper-
ature case was used as the initial microbial concentration for the
case. From the statistical analysis, the parameters A and B in the
Arrhenius model were found more significant than Q0. The squared
sum of fitting residuals were estimated to be 1166 (CFU/mL)2
considering temperature history and 1210 (CFU/mL)2 neglecting it,
which indicated the model accuracy could be improved by
considering the temperature history in determining the model
parameters.
3.3. Temperature dependence of the maximum growth rate
Fig. 3 compares the logarithmic maximum growth rate change
with the inverse of temperature obtained from experiments and
the one calculated using the Arrhenius model in Eq. (8) with pa-
rameters determined during the system identification. The slope of
maximum growth rate in the low-temperature range is steeper,
indicating that it decreases faster with temperature decrease in
Fig. 3. Comparison of the maximum growth rates obtained from experiments, the
one- and the two-zone Arrhenius model fittings.
that range so that a linear fitting using the Arrhenius model fails to
match the change sufficiently.
Guillou and Guespin-Michel (1996) observed the existence of
the two domains of growth temperature in the Arrhenius plot for
the psychrotrophic bacterium, Pseudomonas fluorescens. A similar
trend was also observed in other articles including Labuza and Fu
(1993) and in Mellefont and Ross (2003). Recently Van Derlinden
and Van Impe (2012) evaluated secondary models with a focus on
model performance in the suboptimal temperature range, and they
reported that there was the change temperature, Tc, where the
relation between temperature and the maximum specific growth
altered. They argued that the result revealed a possible short-
coming of the model structure of commonly used secondary
models describing the temperature effect on the microbial growth
rate.
Recalling the observations of Guillou and Guespin-Michel
(1996) as well as Van Derlinden and Van Impe (2012), the rela-
tion between the reciprocal of temperature and the logarithmic
maximum growth rate was assumed as two segments of linear
functions, which is divided at the change temperature, Tc. Although
Van Derlinden and Van Impe (2012) explained the phenomenon as
the impact of cold shock, there is another possibility in this case.
Since there were different microbial species in the packaged milk, it
might be attributed to the result of the different temperature
dependence of the growth rates for different species. The minimi-
zation of SSR was performed with five parameters, A1, B1, B2, Tc, and
Q0, where the frequency factor of the second segment could be
estimated from the continuity equation of the maximum growth
rate at the change temperature Tc as shown in Eq. (13).
   
B B
exp A1  1 ¼ exp A2  2 (13)
Tc Tc
Table 1 shows the determined model parameters. Judging from
the t-values of each parameter, the significance of the model pa-
rameters was in the order of Tc, B2, B1, A1, and Q0. This implies that it
is very important to develop the secondary model to reflect the
experimental observation well. The solid line in Fig. 3 is the mi-
crobial maximum growth rate change with temperature for the
 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540
Table 1
Parameters for the Baranyi and Roberts model coupled with three different secondary models (CI: Confidence Interval, LCI and UCI: Lower and Upper limits of the confidence
interval).
BRA2 BRR1
Value CI
LCI UCI
Q0 1.66  103 4.03  104 2.92  103 Q0
A1 (log(1/hour)) 62.8 60.6 65.1 b (1/hour1/2
C) 2.99  102 2.86  102 3.11  102
B1 (K) 18,450 17,816 19,084 Tmin (
C)
B2 (K) 11,310 11,005 11,615
Tc (
C) 12.1 11.4 12.8
two-zone Arrhenius model determined during the system identi-
fication process, which shows better match with the measurements
than the case of one-zone Arrhenius model. The dotted and solid
lines in Fig. 4(a)e(e) represent comparisons of the fittings of the
Baranyi and Roberts model with one-zone (BRA1) and two-zone
Arrhenius (BRA2) models respectively, and they are compared
with the experimental observations. The y of the y-axis represents
log(N/N0). BRA2 shows better match with experimental observa-
tions than BRA1. BRA1 overestimated the microbial growth for 5
and 30
C conditions where the maximum growth rate was over-
estimated by the model as shown in Fig. 3 while it underestimated
for the other conditions. The two-zone Arrhenius model improved
the match of the maximum growth rate between the fittings and
measurements to yield better fittings of the microbial growth to the
experimental observations. Table 4 lists the accuracy and bias fac-
tors for BRA2. The model accuracy was improved by adding two
more model parameters, i.e., B2 and Tc, and the justification of their
addition into the model set was examined by comparing the values
of BIC between BRA1 and BRA2. They were estimated to be 125 and
71 for BRA1 (p ¼ 3) and BRA2 (p ¼ 5) respectively, where the
number of data n was 196. This indicates that the split of the
temperature range improves greatly the explanatory power of the
model set.
The Baranyi and Roberts model was combined with one- (BRR1)
and two-zone Ratkowsky models (BRR2), and the determined
model parameters are shown in Table 1. BRR1 and BRR2 were
compared against BRA1 and BRA2. The values of BIC for BRR1
(p ¼ 3) and BRR2 (p ¼ 5) were estimated to be 282 and 118
respectively with n ¼ 196. Generally, the Arrhenius model showed
better performance than the Ratkowsky model in this study.
3.4. Effects of an intermediate lag
Fig. 5 compares the predictions of BRA2 for three different
varying temperature history conditions with the measurements.
BRA2 showed measurable overestimations for the first two cases.
Especially for the second condition, the value of bias factor is not
acceptable as shown in Table 2. Although BRA2 showed a good
match for constant temperature conditions, it failed for fluctuating
ones. A possible explanation of the observation was the effect of
intermediate lag. According to Zwietering et al. (1994) and Swinnen
et al. (2004), sudden environmental variations during microbial
growth could lead to delayed growth or an intermediate lag. An
intermediate lag was also observed by Baranyi, Robinson, Kaloti,
and Mackey (1995), which reported a delay compared with the
predictions. Mellefont and Ross (2003) and Swinnen, Bernaerts,
and Gysemans (2005) also described the intermediate lag phase
as a function of the temperature jump. They observed that the in-
termediate lag increased non-linearly with the temperature jump
size, as defined by the pre- and post-shift temperatures. The mi-
crobial growth of the third case shown in Fig. 5(c) seems to be
537
BRR2
Value CI Value CI
LCI UCI LCI UCI
1.08  101 1.34  102 2.03  101 Q0 2.64  103 7.39  104 4.55  103
b1 (1/hour1/2
C) 2.69  102 2.58  102 2.80  102
0.7 0.4 1.0 Tmin.1 (
C) 1.4 1.6 1.1
b2 (1/hour1/2
C) 4.97  102 4.77  102 5.16  102
Tc (
C) 9.5 9.1 9.9
affected by the intermediate lag less due to relatively less number
of temperature shifts.
Swinnen et al. (2004) reported that the Hills and Wright model
could handle the impact of an intermediate lag. The Hills and
Wright model was coupled with the two-zone Arrhenius model
(HWA2) to describe the microbial growth. There are six model
parameters (kn)0, Ea, A1, B1, B2, and Tc in HWA2. Additional data is
needed to determine the model parameter related to intermediate
lag. The data of the first fluctuating temperature condition were
used in this regards. BRA and BRR models used all data from con-
stant temperature conditions (5 data sets) were used to determine
the model constants, and the determined models were validated
for all fluctuating temperature conditions (3 data sets), whereas the
first fluctuating condition data (total 6 data sets) were also included
to estimate model parameters for HWA2 and the remaining two
fluctuating conditions (2 data sets) were used for validation. The
model parameters of Eqs. (5)e(8) and (13) were determined
through the system identification process. The model parameter
(kn)0 in Eq. (7) was found to be statistically insignificant. An alter-
native model was prepared to treat kn as a constant model
parameter. The two model were compared using BIC. Considering
all test cases, the values of BIC were estimated as 185 and 182 for
HWA2 (p ¼ 6) and HWA2 (p ¼ 5) with constant kn respectively with
n ¼ 327. Although SSR for HWA2 (SSR ¼ 509) was lower than that
for HWA2 with constant kn, the penalty of the additional parameter
in HWA2 outweighed the reduction of SSR. The model parameters
for HWA2 with constant kn model are given in Table 3. Table 4
presents the accuracy and bias factors of the model. The data sets
down to the first fluctuating temperature condition were used in
the system identification process, and the last two data set are the
model predictions. The model accuracy for fluctuating conditions
improved while not affecting the performance for the constant
temperature conditions.
Table 5 compares various models considered in this study using
BIC. The model performance should be compared between those in
the same row of the table. The Arrhenius model was preferred over
the Ratkowsky model for the secondary model. While the Baranyi
and Roberts model showed good performance for the constant
temperature cases, the model accuracy could be improved for the
fluctuating temperature cases by adopting the Hills and Wright
model with little penalty for the constant temperature cases.
Finally, the combination of the Hills and Wright model as the pri-
mary model and the two-zone Arrhenius model was selected as the
best one to use for the real-time milk freshness monitoring system.
4. Conclusions
A food freshness assessment model based on microbial growth
and intended for use in an online server system was developed and
successfully applied to commercial low temperature packaged
pasteurized milk. During the development of the model, new issues
538 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540

Fig. 4. Comparison between the model fittings with experimental observations for the cases of set temperature; (a) 5
C, (b) 10
C, (c) 15
C, (d) 20
C, and (e) 30
C.

emerged, including the deviation of the temperature at the center
of the food from the set temperature due to the large thermal ca-
pacity of the packaged milk, and the impact of an intermediate lag.
 The negligence of the deviation of the milk temperature from
the set temperature due to large thermal capacity of food in the
calculation of microbial growth model parameters, could lead to
under- or over-estimation of the growth, and the problem was
resolved by considering the full temperature history of the milk
stored at a constant temperature to determine the model
parameters.
 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540 539

Fig. 5. Comparison of the fittings/predictions made with the Baranyi and Roberts model and the Hills and Wright model coupled with the two-zone Arrhenius model against the
measurements taken under three different fluctuating temperature conditions.

Table 2 Table 3
The accuracy and bias factors, Af and Bf for the Baranyi and Roberts coupled with the Parameters for the Hills and Wright model with the two-zone Arrhenius model with
Arrhenius model under constant temperature conditions and fluctuating tempera- constant kn.
ture conditions.
Parameters Value Confidence interval (95%)
Nominal temperature (
C) BRA2
Lower Upper
Af Bf
kn (1/hour) 7.383  104 2.687  104 1.208  103
5 1.179 0.954 Tc (
C) 10.2 9.6 10.8
10 1.134 0.975 A1 (log(1/hour)) 76.7 70.1 83.3
15 1.106 1.030 B1 (K) 22,430 20,565 24,295
20 1.225 1.134 B2 (K) 12,600 12,231 12,969
30 1.131 1.093
Fluctuating 1 1.205 1.148
Fluctuating 2 1.333 1.299
 Since the squared sum of residuals always decreases with the
Fluctuating 3 1.097 0.932
addition of new model parameter, and using it as the tool to
compare the model prediction performance might end up with
 The predictions of the Baranyi and Roberts model was found to an overfitted model. Since BIC imposes a penalty for the addi-
overestimate the microbial growth of experimental observa- tional parameters in a model, the problem of overfitting could
tions, which was analyzed to be the effect of intermediate lag be avoided by using it. The Bayesian Information Criterion (BIC)
with temperature shift under fluctuating temperature condi- was used as a tool to compare between models, and it could be
tions. The effect of an intermediate lag on microbial growth used as an index to compare the microbial growth models of a
could be considered by adopting the Hills and Wright model. different number of model parameters. The combination of the
The model outperformed the Baranyi and Roberts model, which Hills and Wright model as the primary model and the two-zone
does not consider the intermediate lag. Arrhenius model was selected as the best microbial growth
540 B.-S. Kim et al. / LWT - Food Science and Technology 68 (2016) 532e540

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