Journal of Food Engineering 148 (2015) 4352

Contents lists available at ScienceDirect

Journal of Food Engineering

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

Safety and quality assessment of ready-to-eat pork products in the cold

chain
V. Stahl a, F.T. Ndoye b,, M. El Jabri c, J.F. Le Page c, B. Hezard a, A. Lintz a, A.H. Geeraerd d, G. Alvarez b,
D. Thuault c
a

Arial, Technical Institute for Food Industry, ACTIA centre, 250 rue Laurent Fries, 67412 Illkirch, France
Irstea, Refrigeration Process Engineering Research Unit, 1 rue Pierre-Gilles de Gennes, 92761 Antony, France
ADRIA Dveloppement, Technical Institute for Food Industry, ACTIA centre, Z.A. Creach Gwen, 29196 Quimper cedex, France
d
Division of Mechatronics, Biostatistics and Sensors (MeBioS), BIOSYST, KU Leuven, W. de Croylaan 42, B-3001 Leuven, Belgium
b
c

a r t i c l e

i n f o

Article history:
Received 21 March 2014
Received in revised form 19 September 2014
Accepted 24 September 2014
Available online 5 October 2014
Keywords:
Listeria monocytogenes
Leuconostoc mesenterodes
Lactobacillus sakei
Ready-to-eat pork products
Food safety
Food quality
Cold chain

a b s t r a c t
It is of crucial importance for Ready-To-Eat (RTE) foodstuffs producers to guarantee the quality and safety
of their products under the cold chain variations related to different timetemperature proles. Experimental designs were used to investigate and model the effects of temperature on safety and quality attributes of selected RTE meat products. Three types of RTE sliced pork products (cooked ham, cooked pat
and smoked ham) were stored at different temperatures (5, 8, 12 and 15 C) up to 6 weeks. Microbiological and physico-chemical attributes were followed. Growth parameters of Listeria monocytogenes were
investigated by challenge testing for the three RTE products at the four temperatures. Two lactic acid bacteria (Lactobacillus sakei and Leuconostoc mesenterodes) were also investigated by challenge testing but
only for cooked ham and cooked pat at 8 C. Changes in quality indicators including colour, texture
and water content, water activity and water dripping were evaluated over storage time for the three
RTE products. Spoilage experiments were conducted (at 2, 8, 12, 15 C for 48 days) on cooked ham and
the production of ethanol, as a representative volatile deriving from bacterial metabolism, was correlated
to bacterial outgrowth. Growth parameters of the three strains for the given food were mathematically
modelled and validation tests were performed for L. monocytogenes in cooked ham and cooked pat.
Physico-chemical attributes were not signicantly affected by timetemperature storage. The production
of ethanol on spoiled cooked ham was related to growth of lactic acid bacteria, especially Leuconostoc. A
threshold value of ethanol concentration was dened in relation with a threshold count numbers of LAB
under the conditions studied.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Controlling and improving the quality and safety of chilled
foods at all stages of the cold chain have always been among the
main concerns in order to reduce food losses and health hazards.
Microbial and physico-chemical quality changes may occur in food
products according to their timetemperature history, but also
according to their composition and properties. This is the case for
meat and processed meat products which are ideal for the growth
of spoilage and pathogenic bacteria. Pork meat and Ready-To-Eat
(RTE) pork meals are the main type of meat consumed in Europe
(Mataragas et al., 2008; Verbeke et al., 2010). Moreover, they were
identied as one of the food products where the prevalence of path Corresponding author. Tel.: +33 140966161; fax: +33 140966475.
E-mail address: fatou-toutie.ndoye@irstea.fr (F.T. Ndoye).
http://dx.doi.org/10.1016/j.jfoodeng.2014.09.040
0260-8774/ 2014 Elsevier Ltd. All rights reserved.

ogenic bacteria such as Listeria monocytogenes (L. monocytogenes)

is the highest along the cold chain (EFSA, 2009; Warriner and
Namvar, 2009). This makes the occurrence of L. monocytogenes in
RTE products of particular concern for food business operators
(FBO) and for competent authorities, knowing that the bacterium
poses potential human health risks (EFSA, 2013). In addition to L.
monocytogenes, the pH and high water activity of RTE pork meat
products make possible the growth of other types of bacteria under
refrigerated temperatures (+2/+4 C): the lactic acid bacteria (LAB).
A part of this LAB ora is responsible for spoilage and quality loss
by inducing physico-chemical changes into the food products
(Laursen et al., 2009; Hereu et al., 2012). The physico-chemical
modications are often assessed through quality indicators like
colour, texture, water holding capacity, avour and odour compounds (volatiles organic compounds VOC). Spoilage commonly
manifests itself as off-odours and off-avours due to the presence

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V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

Nomenclature

Latin symbols
a
colour parameter; Hunter redness value ()
aw
water activity ()
b
colour parameter; yellowness value ()
L
colour parameter; lightness value ()
Lag
lag time (h)
N
population size (CFU)
t
time (h)
T
temperature (C)

of VOC. These VOC resulted from bacterial metabolism and their

production is correlated to bacterial cell growth (Leroy et al., 2009).
In this context, assessment of the impact of the food cold chain
on microbial growth and quality attributes of RTE pork meals has
become important in order to improve their shelf life and their
safety. It is also essential to have tools and methods that allow the
quality and safety of foods to be accurately evaluated. Predictive
modelling is one of these tools. Several predictive models have
been developed in order to quantify the effect of various factors
on L. monocytogenes load evolution in various food products
(Rosso et al., 1996; Dalgaard and Vigel Jrgensen, 1998; Zuliani
et al., 2007; Couvert et al., 2010; Mejlholm et al., 2010; Huang,
2013; Polese et al., 2014). These mathematical models were based
on the different phases of bacterial growth: lag; exponential; and
stationary (Buchanan, 1993). They are used through software as
decision support systems for food technologists. Modelling of quality deterioration during food processing and storage has also been
extensively studied (Saguy and Karel, 1980; Labuza, 1984; Van
Boekel, 2008). Kinetic modelling allows the rate of deterioration
reactions in foods to be characterized as a function of temperature.
Rapid assessment of RTE products spoilage can also be achieved by
VOC analysis which could constitute an alternative method to
microbiological analyses.
There are software packages for predictive microbiology available (Couvert et al., 2013; Tenenhaus-Aziza and Ellouze, 2013). In
our knowledge, no such tool has been developed that combines
both safety and quality models of foods. This is one of the objectives of the European FRISBEE project (Food Refrigeration Innovations for Safety, consumers Benet, Environmental impact and
Energy optimisation along the cold chain in Europe) which aims
to provide new tools and concepts for improving refrigeration
technologies. The project has developed novel and innovative
Quality and Energy/Environment Assessment Tools (QEEAT) that
combine food quality and safety together with energy and environmental aspects. These tools were based on mathematical modelling as well as on a knowledge database gathering measured data
of the cold chain performance, among others, in terms of food temperature, and change of quality and safety attributes.
This work is part of the development of QEEAT and focuses on
the following three representative RTE pork meat products:
cooked ham (a RTE cooked meat product in which the original
structure is still recognisable), cooked pt (a RTE cooked and
mixed meat product) and smoked ham (a RTE meat product in
which the original structure is also still recognisable). The objective was to develop kinetic models for safety and quality of the
selected food products. The paper presents the protocols and
experiments implemented in order to develop kinetic models for
bacterial growth and quality attributes changes. It presents also
a potential alternative method to plate counting for detection of
microbial spoilage.

Greek symbols
c
gamma factors ()
l
bacterial growth rate (h1)
Subscripts
max
maximal
min
minimal
opt
optimal
0
initial

2. Materials and methods

Microbial growth experiments were performed in real products
for selected bacterial isolates (L. monocytogenes and LAB ora) as a
function of storage time and temperature. Physico-chemical attributes such as texture, drip-loss and colour were also experimentally determined. In addition, experiments were carried out to
relate volatiles production to microbial population growth according to storage duration and temperature, in order to study a potential, alternative method for detection of microbial spoilage of
products which has the advantage of being faster than classical
plate counting.
2.1. Ready-to-eat pork meals products
Three types of RTE meat products were studied: (i) cooked ham,
(ii) pt, (iii) and smoked ham. They were under modied atmosphere packages (Pothakos et al.) with 50% CO2 + 50% N2 gas
mixture.
2.2. Microbiological study
2.2.1. Bacteria strains
The selected strain of L. monocytogenes (n 352 Arial (F)) was
isolated from environment of a meat industrial plant and was a reference strain of the French program in predictive microbiology:
SymPrevius (Couvert et al., 2010). Two strains were chosen for
LAB, Lactobacillus (Lb.) sakei (n 1322 Aerial) and Leuconostoc mesenterodes (N 74 Aerial), provided from the Aerial collection
(stored at 80 C), isolated from chilled pork meat products.
2.2.2. Optical density measurements
Measurements of optical density allow the cardinal temperature values to be estimated. They describe the effect of temperature on the growth through: (i) the minimal temperature for
growth (Tmin); (ii) the optimal temperature for growth (Topt) and
(iii) the maximal temperature for growth (Tmax) (Neysens and De
Vuyst, 2005). The methodology used was adapted from Membr
et al. (2005) to the two LAB strains using Elliker medium (Biokar)
for incubation. Turbidity growth curves of the strains were generated with a Bioscreen C reader (Labosystemes Honeycomb 2
France). Eleven static temperature levels between 2 C and 40 C
were studied; pH and aw of the medium were kept at optimal levels, namely at 6.8 and at 0.98 respectively.
2.2.3. Microbiological challenge-tests
Challenge tests in the food products were performed for the
strain of L. monocytogenes and for the two strains of LAB, using a
method adapted from Augustin et al. (2011):

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V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

 L. monocytogenes challenge testing was carried out at 5, 8, 12

and 15 C, under modied atmosphere (50% CO2 + 50% N2) in
one batch for each RTE product. One physiological state was
used in this study: cells at the end of the exponential growth
phase. Two subcultures were grown in Brain Heart Infusion
(BHI) at 37 C for 16 h and for 8 h. A third subculture was carried out at 8 C for 6 days, in BHI broth to obtain 9 Log CFU ml1.
Samples of 10 g of product were inoculated with 0.5 ml of the
diluted last subculture to obtain an initial concentration of
2 Log CFU g1 on the surface of the food product. Control
samples without inoculation with L. monocytogenes were also
included. Natural lactic acid ora (1 ml was plated onto de
ManRogosaSharpe (MRS) (Oxoid)); NF V 04-503, and aerobic
microorganisms (1 ml was plated onto PCA (Oxoid), at 30 C for
3 days), pH (Hanna HI 213; NF V04-108), and aw (GBX-FA-St 1;
ISO 21807: 2004) were quantied at 3 days during challenge testing. Hereto, 10 g of the inoculated samples were homogenized
with 90 ml of tryptone salt solution using a stomacher blender.
Ten-fold serial dilutions were carried out and 0.1 ml of the appropriate dilution was plated onto Compass L. monocytogenes (37 C;
48 h) (Biokar): the enumerations of L. monocytogenes were performed on three samples at 11 different times during the lag,
the exponential and the stationary phases of the growth curve.
In total, twelve growth curves of L. monocytogenes in mixedculture (with the potential presence of natural ora) were
obtained.
 LAB challenge-testing were performed with one batch of cooked
ham and one batch of cooked pt under modied atmosphere
(50% CO2 + 50% N2). Three growth curves were obtained at 8 C
in pure culture for each strain and each food product, inoculated
separately. To allow this pure culture tests the food products
were ionized beforehand by 7.5 kGy. Three subcultures of Lb.
sakei 1322 and Lc. mesenterodes 74 strains were grown at
30 C; 30 C and 8 C in Elliker media. Samples of 10 g of product were inoculated with 0.5 ml of the diluted subculture to
obtain an initial concentration of 2 Log CFU g1 in the foodstuff.
Lb. sakei 1322 and Lc. mesenterodes 74 were quantied at regular time intervals to obtain data points representing lag, exponential and stationary phases. The 10 g samples were
homogenized with 90 ml of tryptone salt solution (Oxoid).
Ten-fold serial dilutions were carried out, and 0.1 ml of the
appropriate dilution was plated onto MRS: for the enumeration
of both strains. Control samples without inoculation were also
included. pH and aw were measured.
2.2.4. Mathematical growth modelling of bacteria
2.2.4.1. Cardinal temperature values. Cardinal temperature values of
L. monocytogenes were obtained from Couvert et al. (2010). The
cardinal values of Lc. mesenterodes 74 and Lb. sakei 1322 were estimated from growth kinetics obtained by experimental assays, as
described in Section 2.2.2.
A secondary model that described the inuence of temperature
on the growth rate (Rosso et al., 1995; Pinon et al., 2004) was
employed to estimate the cardinal values of temperature T (Eq.
(1)):
(
TT max TT min 2
for T min < T < T max
cT T opt T min T opt T min TT opt T opt T max T opt T min 2T
0
Otherwise

1
2.2.4.2. Modelling growth kinetics. The growth kinetics in the three
studied RTE meat products were acquired at static levels of temperature (5, 8, 12, 15 C). The evolution of the population size N
as a function of time was described by the logistic model with
delay (Eq. (2)) (Rosso et al., 1996; Pinon et al., 2004). This model

provides a good accuracy using only four descriptive parameters:

lag time (lag), maximum specic growth rate (lmax), initial population size (N0) and the maximum population size (Nmax).

(
lnN

lnN0

for




lmax tlag

1
e
for
lnNmax  ln 1 NNmax
0

t 6 lag
t > lag
2

The differential form of this equation is given as following (Eq.

(3))

8
< dN
0
dt

t 6 lag


: dN lmax  N 1  N
t > lag
dt
Nmax

2.2.4.3. Estimation and validation of growth parameters. The

maximum specic growth rate (lmax) and the lag time (lag) of
L. monocytogenes in obtained at 8 C in cooked ham and cooked
pat, as described above, was used in a next step for model validation purposes. More specically, the optimal growth rate lopt and
the minimal lag time, lagmin, both valid at optimal T, pH and aw conditions, were determined by combining this estimated lmax and lag
phase at 8 C with c(T), c(pH), c(aw) and c(int), the c factors for
temperature, pH, water activity and their interactions (Le Marc
et al., 2002; Augustin et al., 2005). These c factors represent the
impact of these factors on the growth rate lopt and depend on both
the strain and the actual food matrix (Pinon et al., 2004):

lopt
lag

lmax
cTcpHcaw cint

lag min

cTcpHcaw cint

The c(pH), c(aw) and c(int) for both RTE meat products (cooked ham
and cooked pat) are xed for each product and considering the cardinal values of the L. monocytogenes strain (Couvert et al, 2010).
The obtained values for lopt and the minimal lag time, lagmin
were then used to validate the estimated lmax and lag at the others
temperature levels (5, 12, 15 C). These estimated values were
obtained as described under Section 2.2.4.2. It was then compared
to the prediction using the c concept (Eq. (4)). Further, the estimated lag phase was compared to the predicted lag time according
to the following Eq. (6):

lagT

lopt  lag min

lmax T

The Root Mean Squared Error (RMSE) statistical indicator was

used in order to evaluate the difference between the estimated
and predicted values.
2.3. Physico-chemical quality attributes measurements
Evolution of several quality attributes with storage time and
temperature were investigated. Colour, texture, water content,
water activity and water dripping measurements were performed
for each RTE product (pt, cooked ham, smoked ham) depending

Table 1
Cardinal temperature values (T, C) of Lc. mesenterodes 74 and Lb. sakei 1322
estimated in liquid microbiological media.
Cardinal T (C) values

Lc. mesenterodes 74

Lb. sakei 1322

Tmin
Topt
Tmax

0.53
28.04
36.05

0.25
33.02
39.12

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V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

Table 2
Growth parameters of L. monocytogenes estimated at different temperatures in cooked ham, cooked pat and smoked ham, under modied atmosphere, 50% CO2; 50% N2.
T (C)

Log N0 (CFU g1)

Log Nmax (CFU g1)

lag (h)

lmax (h1)

loptd (h1)

lagmin (h)

c(T)

c(pH)

c(aw)

5
8
12
15

2.34
2.51
2.58
2.35

8.56
9.01
9.11
8.67

100
73.92
22.9
12.1

0.0233
0.0361
0.0746
0.103

0.7525

3.55

0.0828

0.9329

0.6212

Cooked patb

5
8
12
15

2.75
2.75
2.75
2.75

8.77
8.39
9.18
9.05

10.56
18.62
8.74
7.87

0.0230
0.0472
0.0974
0.1260

0.7537

1.17

0.0828

0.9574

0.7902

Smoked hamc

5
8
12
15

2.56
2.54
2.61
2.6

2.56
2.54
2.61
2.6

1
1
1
1

0
0
0
0

Product
Cooked ham

a
b
c
d

pH = 6.31 SD = 0.04; aw = 0.980 SD = 0.005.

pH = 6.45 SD = 0.01; aw = 0.986 SD = 0.003.
pH = 5.72 SD = 0.02; aw = 0.969 SD = 0.001.
Calculated from Eq. (4) (Couvert et al., 2010); c(int) = 1.

on the static temperature condition (3, 5, 8, 12 or 15 C) for different storage durations.

2.3.1. Colour measurement
Colour changes were assessed using a colorimeter (CM-2002
Minolta Spectrophotometer Minolta Co., Japan). The CIE Lab colour coordinates (L: lightness value; a: Hunter redness value; b:
yellowness value) were measured. Before measurement, a white
plate (Calibration plate CM-2002) was used to standardize the
instrument under C illuminated condition according to the CIE
(Commission International de lEclairage). The measurements were
replicated three times for each sample at frequent time intervals
during their storage.
2.3.2. Texture measurement
Texture changes were measured by a compression test for pt
and a penetration test for the two ham products using a texture
analyzer (TA-XT2i, Stable Micro Systems, UK) at controlled
temperature.
Compression test: A cylindrical sample of pt (2 cm in height
and 2.5 cm in diameter) was compressed using a cylinder of
25 mm diameter. Constant penetration speed was applied on the
pt until rupture. The cylinder probe approaches the sample at
a speed of 0.2 mm/sec and mashes the sample of pt. A forcedistance curve was obtained and texture parameter (rmness) was
determined as the maximum strength until rupture.
Penetration test: a conical probe was used to penetrate samples
of cooked ham and smoked ham. Constant penetration speed was
applied until perforation. The probe approaches the sample at a
speed of 0.2 mm/sec and penetrates through the slice of ham. A
forcedistance curve was obtained and rmness was also determined as the maximum strength until rupture.

2.3.3. Water content, water activity and water dripping measurements

Water content was measured by sample weighing before and
after being placed in an oven at 103 C; 24 h, at each texture measurement time. Water activity has been measured with an awmeter (Decagon Aqualab 4TE, U.S.) on thin samples, avoiding the
fat to obtain better repeatability in the measurements.
The drip loss measurement may be an easy way to observe a
global effect of alteration in the meat product matrix, allowing a
leaking of fat or water to the outside. To estimate the exudates
of fat and water along storage time, depending on temperature
conditions, the change of weight of food sample before and after
wiping has been measured. The surface of around 200 g of RTE
product (1 slice for the pt and 4 slices for the cooked ham and
the smoked ham) has been wiped with absorbing paper. At each
temperature and time instance, three measurements were realized
on three different slices.
2.4. Volatiles production related to microbial growth
This part of the work was performed in order to investigate the
relationship between the bacterial growth and the production of
some metabolites related to products spoilage. Experiments were
carried out only on cooked ham products (aged 2 weeks), and
stored at temperatures of 2, 8, 12, 15 C during 7 weeks in order
to maximise spoilage. Volatiles production and microbial growth
were analyzed periodically (at 0, 7 14, 29, and 48 days of storage).
2.4.1. Volatiles analysis
Volatiles compounds were analyzed using a static headspace
gas chromatography (SH-GC) with a ame ionization detector
(FID). The chromatograph (PerkinElmer HS 40XL) was equipped
with an autosampler (PerkinElmer Autosystem XL). The capillary

Table 3
Growth parameters of Lb. sakei (Lb. s.) 1322 and Lc. mesenterodes (Lc. m.) 74 obtained at 8 C in pure-culture of cooked ham and cooked pat, under modied atmosphere, 50%
CO2; 50% N2.
Log N0 (CFU g1)

Log Nmax (CFU g1)

lag (h)

lmax (h1)

loptc (h1)

lagmin (h)

c(T)

Cooked ham
Lb. s. 1322
Lc. m. 74

1.8
2.38

8.91
9.07

7.32
1.1112

0.107
0.0896

1.3408
0.6029

0.58
0.17

0.0798
0.1486

Cooked patb
Lb. s. 1322
Lc. m. 74

1.69
2.27

8.67
9.18

37.68
5.34

0.0942
0.0969

1.1804
0.6521

3.0
0.79

0.0798
0.1486

a
b
c

pH = 6.43 SD = 0.04; aw = 0.980 SD = 0.006.

pH = 6.45 SD = 0.01; aw = 0.984 SD = 0.004.
Calculated from lmax = lopt  c(T); c(int) = 1.

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V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

Table 4
Validation of estimation growth parameters at the three temperatures level (5 C, 12 C and 15 C) for cooked ham and cooked pat Root Mean Squared Errors (RMSE) indicator
results.
T (C)
Cooked ham
5
12
15

Cooked patb
5
12
15

a
b

Estimated lmax (h1)

Predicted lmax (h1)

Estimated lag (h)

Predicted lag (h)

0.0233
0.0746
0.1030

0.0189
0.0683
0.0990

100.30
22.90
12.07

95.99
38.01
26.16

RMSE(lmax)

0.005

RMSE(lag)

12.18

0.0230
0.0974
0.1260

0.0240
0.0891
0.1292

RMSE(lmax)

0.005

10.56
8.74
7.87
RMSE(lag)

38.22
9.02
6.98
15.98

1

Parameters used: lopt = 0.75 (h ) lagmin = 3.55 (h).

Parameters used: lopt = 0.75 (h1) lagmin = 1.17 (h).

column used was a PE-WAX (PerkinElmer) with a length of 30 m,

an internal diameter of 0.32 mm and a lm thickness of 0.25 lm.
The carrier gas used was helium at a pressure of 200 kPa. Cooked
ham samples were grinded with 20% of NaCl and 7 g of the mixture
were transferred into a 20 ml headspace vial. Samples were equilibrated at 50 C for 45 min in the SH oven prior to injection. The
needle was held at 150 C and the transfer tube at 170 C. A volume
of 600 ll was taken in the headspace of the vial and injected in the
GC in splitless mode. The oven temperature program of the GC was
set: an initial step at 40 C for 10 min, a linear increase from 40 to
225 C at 10 C/min and a nal step at 225 C for 5 min.
The main detected peaks were identied by comparison with
retention time of standard compounds: ethanol, acetic acid,
3-methyl butanol (Sigma Aldrich). No calibration curve was constructed for the concentration of metabolites, due to the complexity of the cooked ham matrix. Also, no internal standard was used
thanks to the good results in terms of accuracy and precision of FID
as detection system, in comparison with mass spectrometry
(where the use of internal standard is necessary to correct instrumental variability) (Su et al., 2008; Bronsema et al., 2012). So, the
peak areas were used to quantify the relative changes in volatiles
concentration over time. Analyses were performed in triplicate
for each temperature and each time instance.
2.4.2. Bacterial enumeration
The following microbial populations were enumerated: total
lactic acid bacteria (Labuza), Brochothrix thermosphacta, Leuconostoc, Enterobacteriaceae. At each sampling moment, 10 g of cooked
ham were diluted in saline solutions and homogenized for 1 min
in a Stomacher. Aliquots (1 ml) of the appropriate decimal dilutions in saline solutions were used to prepare spread plates for
the enumeration on selected media and incubation conditions:
MRS (pH 5 and 7), 30 C for 3 days and 15 C for 7 days for total
LAB; Streptomycin Thallous acetate actidione agar (Oxoid) at
25 C for 3 days for Br. thermosphacta; Leucocan agar (Aerial) at
30 C for 3 days for Leuconostoc; Violet Red Bile Glucose (Oxoid)
at 37 C for 3 days for Enterobacteriaceae.
3. Results and discussion
3.1. Bacterial kinetic models of chilled RTE pork meals
Studies have been directed towards developing models to
describe the combined effects of environmental factors on microbial
growth of pathogens in meat products (Mataragas et al., 2010;
Mejlholm et al., 2010). The effect of factors (pH, aw, T, and NaNO2)
was investigated by developing predictive models for the lag phase
and the maximum specic growth rate of strains of naturally present

spoilage microorganisms in meat products (Devlieghere et al., 1998;

Koutsoumanis et al., 2006). In this study, one specic strain of
L. monocytogenes and two strains, Lc. mesenterodes and Lb. sakei
were used as test organisms.
3.1.1. Cardinal temperature values
Table 1 shows cardinal temperature values of Lc. mesenterodes
74 and Lb. sakei 1322 estimated in our study. Cardinal temperature
values were estimated from experimental growth kinetics acquired
in liquid microbiological media. Results obtained agree with the
psychrotrophic character of these microorganisms and their ability
to grow at refrigeration temperatures. These results are in line with
cardinal values of LAB reported in the literature (Wijtzes et al.,
1998; Ellouze et al., 2008; Ellouze and Augustin, 2010).
3.1.2. Estimation of bacterial growth parameters in RTE meat products
The results of the growth parameters of L. monocytogenes on
the three products studied are shown in Table 2. A signicant
increase in the concentration of L. monocytogenes at each temperature studied (5, 8, 12 and 15 C) was observed for cooked ham
and pt, while no growth was observed for smoked ham samples. Initial pH and aw values of the three RTE pork meals (see
on Table 2) classify the products in the food category allowing
growth of L. monocytogenes and these parameters did not signicantly evolve during the challenge tests. MAP Packaged cooked
ham slices were identied as supporting well the growth of
pathogens (Uyttendaele et al., 1999; Koutsoumanis and
Angelidis, 2007; Uyttendaele et al., 2009; Mejlholm et al., 2010;
Augustin et al., 2011). The absence of growth of L. monocytogenes
on smoked ham may be explained by an inhibition by LAB ora
(Cornu et al., 2011). The high initial levels (8.5 Log CFU g1) of
natural LAB population which were found in the smoked ham
at the onset of the experiments suggest a competitive effect.
These high levels of LAB can be linked to several processing steps
(maturation, smoking and drying) without heat treatment. These
microora were naturally present on cooked ham and cooked
pat, but initially at much lower levels (below the detection limit
value of 0.3 Log CFU g1). Hence, they did not signicantly affect
the growth potential of L. monocytogenes, as a maximum level
above 8 Log CFU g1 was reached only at the end of storage.
The smoked ham is actually in the pH and aw range (Table 2) of
deli salads, were mostly no growth was observed. lopt of cooked
ham and pt (Table 2) are consistent with those obtained by
Ellouze and Augustin (2010).
Table 3 shows the results of growth parameters of Lb. sakei 1322
and Lc. mesenterodes 74 at 8 C in pure culture in cooked ham and
pt. lopt is the medium and microbe specic parameter. Hence, it
was calculated according to the equation, lmax = lopt c(T).

48

V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

Table 5
Water content and water activity of cooked ham, cooked pt and smoked ham as a function of storage temperature at the start (day 0) and at the end of storage.
Product

Day

Cooked ham

Initial
Enda

Cooked pt

Initial
Endb

Smoked ham

a
b
c

Initial
Endc

T (C)

Water content (%)

Water activity

3
8
12
15

73.2 0.3
74.2 0.1
73.5 0.3
72.5 0.5
73.3 0.3

0.983 0.003
0.982 0.001
0.982 0.001
0.981 0.001
0.979 0.002

3
8
12
15

55.9 1.2
55.1 2
53.9 0.5
55.8 1.9
55.8 1.1

0.973 0.002
0.977 0.002
0.973 0.002
0.974 0.002
0.970 0.007

3
8
12
15

53.4 0.5
49.6 0.5
52.9 0.7
52.7 0.9
53.6 0.2

0.902 0.008
0.896 0.009
0.905 0.012
0.906 0.07
0.904 0.07

End day = 32.

End day = 24.
End day = 34.

200000

1,6
3C
8C
12C
15C

180000
160000

1,2

Peak area (V*sec)

Drip loss (g/100g of product)

1,4

1
0,8
0,6
0,4

140000
120000
100000
80000
60000
2C

40000

8C

0,2

20000

12C
15C

0
0

10

15

20

25

30

35

Time (days)
Fig. 1. Variations of drip loss of cooked ham with storage time and temperature.

Products may incorporate a variety of microorganisms that continuously compete and interact during storage. In the present study,
we aimed to evaluate the kinetics of individually inoculated strains
of lactic acid bacteria. Hence the estimated growth parameters (i.e.,
growth rate, lag time) are not including any potential interactions
that may occur between different naturally present LAB species.
The concentration of the two strains increases during the period
of the challenge tests. The environmental conditions (pH, aw) of
both RTE products permit the growth of both strains. The maximum
levels reached were above 8 Log CFU g1. Garca-Gimeno et al.
(2005) studied combined effect of T, pH level (5.57.5), sodium
chloride and sodium nitrite levels on the specic growth rate, lag
time of Lc. mesenterodes in modied MRS medium. At quite similar
conditions (10.5 C; pH 6.5) growth rate and lag duration were
respectively 0.16 h1 and 11.39 h. The derived growth kinetic
parameters of observed growth curves of spoilage LAB in naturally
contaminated meat product were, at 8 C, 15.84 h1 and 40.32 h
(Mataragas et al., 2006). Devlieghere et al. (1998) studied growth
rate of Lb. sakei at 10 C: it was signicantly higher in modied
MRS (lmax of 0.308 h1; lag of 6.2 h) than in cooked ham (lmax of
0.117 h1; lag of 2.5 h) and BHI (lmax of 0.111 h1; lag of 4.3 h).
3.1.3. Validation of the bacterial kinetic model
Optimal growth rate and lag phase (lopt and lagmin) of L. monocytogenes, based on the result at 8 C, were used to validate the

10

20

30

40

50

60

Time (days)

Fig. 2. Spoilage experiments with cooked ham response surfaces of ethanol

obtained at SH-GC during storage at different temperatures.

estimation of lmax and lag phase at 5, 12 and 15 C for cooked

ham and cooked pat (validation was not done on smoked ham
because of the absence of L. monocytogenes growth during the challenge tests). Table 4 displays estimated and predicted values of
lmax and lag obtained for cooked ham and cooked pat, together
with the RMSE indicators. There is no signicant difference
between estimated and predicted values of lmax for both RTE products. The RMSE indicator shows a value of 0.5% for both cooked
ham and cooked pat. Baranyi and Roberts (1995) reported standard errors of 10% of the estimated growth rate (lmax) in synthetic
media. In order to describe the variability of lmax, Augustin et al.
(2011) used the residual variances corresponding to a coefcient
of variation of 20%, when performing a challenge test for a given
batch and a given physiological state. With regard to the lag time
results, some differences are observed between estimated and predicted lag times, mainly in the case of cooked pat (Table 4). For lag
time, the prediction is larger than the estimated one and therefore
unsafe. The simulation of the lag time is complex in predictive
microbiology as it depends on many factors (Swinnen et al.,
2004; Ferrier et al., 2013). The estimations of lopt and lagmin with
at temperature of 8 C allowed the estimation of the growth
parameters in other temperature conditions. Concerning Lb. sakei
and Lc. mesentroides, only the temperature of 8 C was considered
in this study.

49

V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

Table 6
Spoilage experiments with cooked ham cell counts (in Log CFU g1) of several microorganisms as a function of temperature and duration of storage.
Storage temperature

2 C

8 C

12 C

15 C

Day

14

29

48

14

29

48

14

29

48

14

29

48

LAB 30 Ca
LAB 15 Ca
Leuconostoc
Br. thermosphacta
Enterobacteriaceae
Yeast and moulds

1.3
1.3
3.5
5.0
1.3
1.3

1.3
1.3
1.3
6.3
1.3
1.8

1.3
1.3
1.3
5.8
1.3
3.0

1.3
1.3
1.3
6.6
1.3
1.3

8.9
8.8
8.7
5.8
1.3
5.6

7.9
8.3
8.7
5.3
1.3
3.5

6.7
6.8
9.1
5.5
1.3
3.2

8.9
9.1
9.0
7.1
1.3
6.4

8.6
8.5
8.8
6.5
1.3
3.8

7.5
7.4
9.0
5.2
1.3
1.3

5.1
1.3
8.9
6.4
1.3
2.8

1.3
1.3
1.3
6.4
1.3
4.3

8.2
8.4
8.4
5.2
1.3
3.0

8.9
8.8
8.9
6.6
1.3
3.1

4.5
1.3
9.0
4.9
1.3
1.6

8.9
8.9
8.8
5.0
1.3
1.3

8.8
8.7
8.9
5.3
1.3
1.3

Temperature of incubation for enumeration.

3.2. Physico-chemical quality changes

3.2.1. Effect of storage on colour stability
Colour was quantied by measuring the variation of L, a and b
values as a function of storage temperature and duration. No signicant changes were observed for the three colour parameters
throughout the storage time and for the four temperatures tested
(Statistical analysis, StatGraphics Plus: L: p = 0.22; a: p = 0.31;
b: p = 0.26 for cooked ham, similar results were obtained for
pt and smoked ham). These results agreed with the ndings of
Garca-Esteban et al. (2004) who reported no colour changes in
MAP sliced ham after 8 weeks of storage at 4 C. Mller et al.
(2000) also reported similar conclusions of MAP sliced ham at
5 C during 27 days. Discolouration of meat products is often
attributed to oxidative processes of myoglobin. In MAP meat products, oxidation processes are due to the interactions between the
oxygen level in the package, the product to headspace volume ratio
and the light intensity (Mller et al., 2003; Nannerup et al., 2004).
The absence of discolouration for the RTE samples tested in this
work may be explained by the low level of oxygen in the package.
These results suggested that colour parameter is not really a relevant quality indicator in these cases studies.
3.2.2. Effect of storage on texture characteristics
Texture proles of the three RTE pork meals were assessed by
measuring the rmness values throughout the storage time and
for the four temperatures tested. The texture parameter was found
to be relatively constant: cooked ham: p = 0.14; pt: p = 0.15;
smoked ham: p = 0.62. In agreement with these results, Jeremiah
et al. (1992) and Garca-Esteban et al. (2004) reported no signicant difference in texture prole and in water content of MAP
sliced ham at 4 C stored during 8 weeks. Cilla et al. (2006) have
studied texture changes of dry-cured ham stored at 4 C for
8 months under MAP. They emphasized that textural changes were
statistically signicant between the 6th and the 8th months. As it
was stated above for colour attributes, these results showed that
texture parameters are not either a relevant quality indicator for
the type of RTE products and storage conditions studied, in agreement with Mller et al. (2003) and Nannerup et al. (2004).
3.2.3. Effect of storage on water components
Table 5 presents starting and end values of water content and
water activity for the three RTE products at the different storage
temperatures studied. For all conditions tested, no signicant differences were observed in water content of RTE pork products.
Same observations were made for the aw values. Drip loss change
depends greatly on the water content, and the water binding properties of the meat products matrix. The greater loss appears for the
cooked ham, which is obvious due to its higher water content and
aw (Fig. 1). At each sampling moment, a new sample, stored from
the beginning was being measured. In the case of the cooked
ham a cumulative loss lower than 3 g in the most unfavorable conditions of storage (15 C) is observed, which is still not relevant in
comparison with the 200 g of studied product.

At the 4 temperatures, for the smoked ham (at 3 C,

Yield = 0.07 0.04%; at 8 C, Yield = 0.24 0.14%; at 12 C,
Yield = 0.54 0.94%; at 15 C, Yield = 0.23 0.11%) and the pt
(at 3 C, Yield = 0.11 0.03%; at 8 C, Yield = 0.15 0.01%; at
12 C, Yield = 0.11 0.03%; at 15 C, Yield = 0.15 0.04%), similar
values were obtained, even lower, <0.3 g and <0.4 g respectively.
3.3. Production of volatiles and correlation with the microora
Bacterial spoilage of meat products was largely associated with
off-odours and off-avours due to volatiles production resulting
from bacterial metabolism. Several studies have been dedicated
to the identication of these volatiles compounds and their specic
role in producing a given off-odour and/or off-avour (Stanley
et al., 1981; Dainty et al., 1989; Borch et al., 1996; Dainty, 1996;
Huis int Veld, 1996; Gram et al., 2002; Pin et al., 2002; Mayr
et al., 2003). In this study, the relationship between the development of volatile components and the growth of spoilage bacteria
in cooked ham as a function of temperature and time was investigated. Linking these two parameters could allow the development
of a fast method for the determination of the microbial spoilage
status of products.
3.3.1. Volatile compounds
The inuence of storage temperature and time (2, 8, 12, 15 C
during 7 weeks) on the evolution of volatiles in cooked ham was
assessed by SH-GC. Particular attention was given to the detection
of volatile compounds, originating from bacterial spoilage process.
Several peaks were obtained in the chromatograms, but unfortunately, only ethanol was clearly identied thanks to preliminary
calibrations. Thus, the study was focused on ethanol, which also
gave the highest response surface. Otherwise, it was recognized
that ethanol can result from the carbohydrate metabolism of LAB
(Kandler, 1983; Leroy et al., 2009). Fig. 2 shows the evolution of
the surface of peak response of ethanol as a function of storage
time and temperature. The production of ethanol increased as storage temperature and duration increased. The increase with time
was by a factor of about 9, 16, 32 and 35 respectively at 2, 8 12
and 15 C between the beginning and the end of storage time
(48 days). This increase is very fast during the rst days of storage,
but peak areas remained fairly stable beyond 16 days, for all temperatures. Leroy et al (2009) studied the production of volatiles in
stored (4, 7, 12, 26 C for 7 weeks) MAP ham samples. They
reported that ethanol was among the compounds which give the
highest responses as volatile compounds deriving from bacterial
metabolism. They also mentioned that ethanol production was faster as temperature increased, yet they did not nd a stabilisation of
the ethanol production at the end of the storage time.
3.3.2. Microbial growth
For the spoilage experiments with cooked ham, microbial enumeration was carried out in parallel to volatiles analysis. Table 6
shows the results of cell counts for the microbial populations
investigated at 2, 8, 12 and 15 C for 0, 7, 14, 29 and 48 days of stor-

V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

(b) 200000

10

180000

160000

160000

140000

140000

120000

120000

100000

80000

100000

Ethanol
Br. thermosphacta
Total LAB
Leuconostoc

80000
60000

4
3

40000

20000

Peak area (V*sec)

10

20

30

40

50

3
Ethanol
Br. thermosphacta
Total LAB
Leuconostoc

40000
20000
0

60000

60

10

20

Time (days)

140000
120000
100000
80000
60000
Ethanol
Br. thermosphacta
Total LAB
Leuconostoc

40000
20000
0
30

40

50

60

Time (days)

Peak area (V*sec)

Peak area (V/sec)

160000

20

40

50

1
0
60

(d) 200000

Log CFU/g

10
9
8
7
6
5
4
3
2
1
0

180000

10

30

Time (days)

(c) 200000

Log CFU/g

10

180000

Log CFU/g

Peak area (V*sec)

(a) 200000

10

180000

160000

140000

120000

100000

80000

Log CFU/g

50

60000
Ethanol
Br. thermosphacta
Total LAB
Leuconostoc

40000
20000
0
0

10

20

30

40

50

2
1
0
60

Time (days)

Fig. 3. Production of ethanol related to the growth of total LAB, of Leuconostoc and Br. thermosphacta in cooked ham as a function of time at (a) 2 C, (b) 8 C, (c) 12 C and (d)
15 C.

age. The detection limit of the method was at 1.3 Log CFU g1. At
day 0, the culturable bacterial population mainly consisted of Br.
thermosphacta with a total count of 5 Log CFU g1. The composition
of the ora of the cooked ham was modied during storage. More
specically, the level of Br. thermosphacta stayed constant while
total LAB, in particular Leuconostoc, became dominant during storage time. The population density reached a maximum of 89 Log
CFU g1 after 48 days of storage at 2 C and after 29 days at 8 C
and 15 C. At 12 C and 29 days, the level of LAB was below the
threshold of detection. The process of cooking has been demonstrated to be sufciently destructive to the Gram-positive and
Gram-negative foodborne pathogens and major background ora
like LAB. However, industrially-produced cooked ham is exposed
to the post-cooking environment and is handled prior to nal packaging. The post-cooking contamination of the cooked ham packs
after heat treatment is heterogeneous. Incubation at 30 C or
15 C of MRS agar did not make a difference in the obtained value
for the total LAB.
Vasilopoulos et al. (2008) reported same variation range of Br.
thermosphacta counts (between 4 and 7 Log CFU g1) for spoilage
experiments in MAP artisan-type sliced cooked ham incubated at
4, 7, 12, 26 C during respectively 57, 56, 35 and 24 days. They
found that Br. thermosphacta were dominant at the initial day of
the spoilage experiments but their counts decreased towards the
end of the experiments due to competition by LAB. In this study,
total LAB and Leuconostoc did not develop at low temperature
(2 C), except after 48 days of storage, but a count number exceeding 8 Log CFU g1 was detected at 8 C, 12 C and 15 C just after
1 week of storage. Similar results were published by Vasilopoulos
et al. (2008) who found that Lc. carnosum represented 9171% of
the total MRS agar population from 4 to 12 C. Enterobacteriaceae
was not detectable in our study.
The dened spoilage reference LAB levels exceeding 6 Log
CFU g1 was suggested by Vasilopoulos et al. (2008). Others
authors set this level at 7 Log CFU g1 for cooked sliced ham under
chilled storage conditions (Ruiz-Capillas et al., 2007; Slongo et al.,
2009). Mataragas et al. (2006) and Kreyenschmidt et al. (2010)

published a threshold level of 8 Log CFU g1 for cooked meats at

4 C. Pothakos et al. (2012) observed quality deterioration for vacuum packaged ham slices at the end of shelf life, with LAB counts
on MRS (incubated at 30 C) of only 5.36 Log CFU g1, corresponding to counts on MRS, incubated at 22 C, of 7.78 Log CFU g1. In
general, it is known that it is not possible to dene a unique level
of LAB counts that would correspond to spoilage for all conditions
and all type of perishable food products: the quality deterioration
depends on the ecosystem of raw materials, the specic spoilage
organism (if applicable), the initial proportion of spoilers in the
total ora and their metabolic activity, recipe and process parameters, storage temperature level, composition of atmosphere and
the level of hygienic control (Laursen et al., 2009).
3.3.3. Correlation of volatiles production with LAB growth
The production of ethanol compound was paralleled to the
growth of LAB, Br. thermosphacta and Leuconostoc in Fig. 3ad. A
relationship between the increase of LAB and Leuconostoc and the
production of ethanol can be evidenced: for all storage temperatures, the peak area increased with the growth and remained stable over time at the end of the active growth phase. This
observation indicated that production of ethanol is linked to the
growing cells of LAB and of Leuconostoc in particular. Leroy et al.
(2009) have reported a correlation between ethanol production
and growing cells in spoiled, artisan-type, MAP cooked ham. In
addition to the production by active growing cells they also
observed a production by already present cells, contrary to our
results. Mayr et al. (2003) obtained a strong correlation between
the production of ethanol and the numbers of LAB in meat spoilage
experiments. They reported that LAB are metabolically active
under microaerophilic conditions and produce ethanol besides lactic acid. Vasilopoulos et al. (2010) found, in a co-culture of Br. thermosphacta and Lc. carnosum in MAP artisan-type cooked ham, that
ethanol and lactic acid were formed in equal amounts from glucose. The production of ethanol seems to be a good indicator of
high bacterial growth of MAP cooked ham under the uctuations
of temperatures in the cold chain, even if several others volatiles

V. Stahl et al. / Journal of Food Engineering 148 (2015) 4352

such as 3-methyl butanol, acetic acid and acetoin were also identied to be representative of LAB growth in meat products (Borch
et al., 1996; Pin et al., 2002; Mayr et al., 2003; Leroy et al., 2009).
Using the growth curves of LAB and the evolution of production
of ethanol as shown on Figs. 2 and 3, threshold concentration of
ethanol can be dened in relation with the count numbers of
LAB (6 Log CFU g1). Values of 43 000, 35 000, 25 000 and 22
000 lV sec were found respectively at 2, 8, 12 and 15 C respectively after 40, 5, 3 and 2 days of storage. However, it should be
kept in mind that these values are valid in the specic case of this
study, as the heterogeneity of products make difcult the development of quality indices reliable for all RTE meat products (Laursen
et al., 2009).
4. Conclusion
Food business operators producing Ready-To-Eat (RTE) foodstuffs must be able to demonstrate that the products will comply
to regulatory specications in terms of food safety. At the same
time, various food quality aspects are also important to ensure
the economic position of the producers. It is obvious that the actual
timetemperature proles a food product undergoes are of paramount importance for guaranteeing food safety and quality. Cardinal values of temperature for two lactic acid strains, Lb. sakei and
Lc. mesenterodes were estimated, and growth parameters of L.
monocytogenes and both lactic acid strains on three representative
RTE cooked meat products (cooked ham, cooked pt, and smoked
ham) were identied. Physico-chemical food attributes were not
signicantly affected by time/temperature storage in our study.
The relationship between the production of a volatile component,
ethanol, and the growth of spoilage bacteria population in cooked
ham as a function of temperature and time were assessed and
strong parallels have been found. However, variations between
batches, processing parameters and spoilage require identication
of the relevant case-specic indicators useful for the control of
microbial spoilage of processed meat products.
Acknowledgments
The research leading to these results has received funding from
the European Communitys Seventh Framework Programme (FP7/
2007-2013) under Grant Agreement No. 245288, coordinated by
Irstea (Anthony, France). The authors are thankful to the SymPrevius consortium (www.symprevius.org) for making available the L.
monocytogenes strain used for this study.
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