2313

Journal of Food Protection, Vol. 70, No. 10, 2007, Pages 2313–2320
Copyright 䊚, International Association for Food Protection

Effect of Packaging and Storage Temperature on the Survival of

Listeria monocytogenes Inoculated Postprocessing on
Sliced Salami
ANTONIA S. GOUNADAKI,1 PANAGIOTIS N. SKANDAMIS,1,2* ELEFTHERIOS H. DROSINOS,2 AND
GEORGE-JOHN E. NYCHAS1

1Laboratory of Microbiology and Biotechnology of Foods and 2Laboratory of Food Hygiene and Quality Control, Agricultural University of Athens,
Department of Food Science & Technology, Iera Odos 75, Athens, 11855, Greece

Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020

MS 06-658: Received 20 December 2006/Accepted 6 May 2007

ABSTRACT
The survival of postprocess Listeria monocytogenes contamination on sliced salami, stored under the temperatures as-
sociated with retail and domestic storage, was investigated. Sliced salami was inoculated with low and high concentrations of
L. monocytogenes before being packaged under vacuum or air. Survival of L. monocytogenes was determined after storage of
sausages for 45 or 90 days for low or high sample inocula, respectively, at 5, 15, and 25⬚C. All survival curves of L.
monocytogenes were characterized by an initial rapid inactivation within the first days of storage, followed by a second, slower
inactivation phase or ‘‘tailing.’’ Greater reduction of L. monocytogenes was observed at the high storage temperature (25⬚C),
followed by ambient (15⬚C) and chill (5⬚C) storage conditions. Moreover, vacuum packaging resulted in a slower destruction
of L. monocytogenes than air packaging, and this effect increased as storage temperature decreased. Although L. monocytogenes
numbers decreased to undetectable levels by the end of the storage period, the time (in days) needed for this reduction and
for the total elimination of the pathogen decreased with high temperature, aerobic storage, and high inoculum. Results of this
study clearly indicated that the kinetics of L. monocytogenes were highly dependent on the interaction of factors such as
storage temperature, packaging conditions, and initial level of contamination (inoculum). These results may contribute to the
exposure assessment of quantitative microbial risk assessment and to the establishment of storage-packaging recommendations
of fermented sausages.

Listeria monocytogenes is a psychrotrophic microor-
ganism that is ubiquitous in nature and common in foods
of both plant and animal origin. It is recognized as a po-
tential contaminant of ready-to-eat (RTE) products, as sev-
eral outbreaks of L. monocytogenes were found to be as-
sociated with the consumption of refrigerated RTE products
(7, 8). The high mortality rate (30%) associated with lis-
teriosis has caused L. monocytogenes to be considered a
public health hazard (40). However, the high incidence of
this microorganism (18) and its ability to persist within the
food-processing environment for years (31) make control
of L. monocytogenes a challenge for the food industry.
Fermented sausages such as salami are widely popular
deli meat products all over the world. Their sales have sig-
nificantly increased in the last two decades on both sides
of the Atlantic Ocean. They are consumed as appetizers
(RTE), side dishes, dips, or sandwich fillers, and they are
purchased as packed retail products or per weight through
the deli section of supermarkets, delicatessen stores, or food
service operations. They are considered one of the safest
food groups for humans because their manufacture involves
a combination of hurdles (low pH, low water activity, pres-
ence of competitive microflora, and curing salts) to patho-
gen survival or growth. However, studies have shown that
* Author for correspondence. Tel: ⫹302105294684; Fax: ⫹302105294683;
E-mail: pskan@aua.gr.
foodborne pathogens such as L. monocytogenes may over-
come these hurdles in French sausages (38), chorizo sau-
sages (20), and Italian-style salami (32). Although L. mono-
cytogenes is among the most frequently detected pathogens
in fermented sausages, reaching prevalence levels of up to
30% (2, 27), clinical listeriosis cases have yet to be linked
to the consumption of fermented sausages contaminated
with L. monocytogenes (32). So far, there is only one epi-
demiologic association of L. monocytogenes with salami in
Philadelphia, but no confirmed outbreak due to the con-
sumption of such products with Listeria has, to our knowl-
edge, been reported (29). However, salami belongs to the
category of commercially available RTE meat products for
which the U.S. Food and Drug Administration–Center for
Food Safety and Applied Nutrition (40) has clearly outlined
the need for further research related to survival and growth
kinetic data of L. monocytogenes.
The intrinsic characteristics (e.g., low pH, water activ-
ity) of fermented sausages, as well as the refrigeration tem-
peratures, have been employed as fundamental strategies
for their preservation (39). In addition, dry fermented sau-
sages of water activity ⬍0.90 and pH of approximately 4.5
(14) may be stored at ambient temperatures. Indeed, prelim-
inary studies in our laboratory regarding the preferences
and handling habits of European Union consumers on fer-
mented sausages show that 40.2% of consumers store these
2314 GOUNADAKI ET AL.
products at room temperature (25⬚C in kitchen or 15⬚C in
cellar) (14). However, the risk associated with the survival
of L. monocytogenes is likely to differ among fermented
sausages, mainly because of the complexity and variety of
manufacturing recipes used. Thus, there is a need for sys-
tematic scientific knowledge about the safety of such foods.
For marketing and product quality reasons, nowadays
fermented sausages are usually vacuum packaged and
stored, distributed, and displayed at refrigeration tempera-
ture (40), conditions that positively affect their appearance
and shelf life. According to a preliminary survey on con-
sumer preferences for dry fermented sausages, the majority
of consumers prefer to buy these RTE products as whole
sausages, while an increasing percentage of consumers pur-
chase them sliced and vacuum packaged either from the
processing plants or at retail points at the time of sale (14).
Before consumption, the majority of the consumers (81.1%)
repack these products in plastic (bags) (14). Although there
is excessive documentation on the fate of L. monocytogenes
on vacuum-packaged fermented sausages (9, 10, 19), there
is limited information on the comparative evaluation of the
survival of pathogens under air or vacuum. Thus, there is
a need for data related to the behavior of L. monocytogenes
in deli meat products packaged under vacuum or unpacked.
Such data are also of value for the implementation of mi-
crobiological criteria for foods illustrated by European
Union Regulation 2073/2005 (11). Therefore, the objective
of the present study was to evaluate the effect of packaging
under air or vacuum on the survival of postprocessing L.
monocytogenes contamination on slices of dry fermented
salami stored at 5, 15, and 25⬚C.
MATERIALS AND METHODS
Preparation of bacterial inoculum. L. monocytogenes Scott
A was used for the inoculation of sliced salami (kindly provided
by Dr. Eddy Smid, Agricultural Research Institute, Wageningen,
The Netherlands). This strain was selected because it is often used
in low temperature or acid challenge studies because of its clinical
origin and the strong epidemiologic association of serotype 4b
with human listeriosis (28). Furthermore, the use of individual L.
monocytogenes strains of various origin, as applied by several
researchers (13, 25, 30, 35, 43), may provide representative data
equal to those of studies with multiple clinical or outbreak iso-
lates. The stock culture was activated from frozen beads (Protect,
Technical Service Consultants Ltd., Lancashire, UK) in 10 ml of
tryptone soy broth (TSB; LAB M, International Diagnostics
Group Plc., Bury, UK) for 18 h at 30⬚C. A 0.1-ml portion of the
activated culture was transferred to 100 ml of TSB and incubated
at 30⬚C for 18 h. Bacteria were harvested after centrifugation at
4,000 ⫻ g for 10 min at 4⬚C. The cell pellet was washed and
resuspended twice in 100 ml of 0.85% saline before inoculation.
Preparation of salami. Sliced salami used in this study was
obtained from a commercial sausage manufacturing company. The
salami was processed on a meat slicer preset to yield 6- to 7-g
slices and transferred to the laboratory for surface inoculation. The
upper surface of each slice was inoculated with 0.025 ml of the
L. monocytogenes culture by spreading the inoculum over the en-
tire surface with a sterile bent glass rod. Following a 10-min rest-
ing period for bacterial attachment, the slices were flipped over,
and the other side was inoculated. Six to eight of the inoculated
J. Food Prot., Vol. 70, No. 10
slices (50 g) were placed into clear polyethylene bags, packaged
under air or vacuum, and stored at 5, 15, or 25⬚C. Samples were
stored for 45 or 90 days for low or high inoculum level, respec-
tively.
Vacuum packages were performed with a Henkovac 1900
(Hertogenbosch, The Netherlands). For samples stored under air,
the Ziploc-type bags were fastened about 0.5 cm from the top to
achieve an unoccupied volume of 650 ml. Two different batches
of salami, within a 3-month period (batch A and batch B), were
prepared under the same manufacturing conditions. Both batches
were individually inoculated as described above, with different
inoculum levels, which resulted in a low (batch A, 4.6 log CFU/g)
and a high (batch B, 6.5 log CFU/g) population density of L.
monocytogenes. The above procedures were performed twice, and
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020
the samples were analyzed in duplicate.
Microbiological analysis. At specific time intervals (0, 3, 6,
10, 13, 17, 20, 24, 27, 31, 38, and 45 days of storage for batch
A and 0, 3, 6, 10, 13, 17, 20, 24, 27, 31, 38, 45, 58, and 90 days
of storage for batch B), two packages were removed from storage
and analyzed independently. Specifically, from each package, 25-g
portions of salami were aseptically transferred to a stomacher bag
containing 225 ml of sterile 0.1% peptone water with salt (NaCl,
0.85%, wt/vol) and macerated for 1 min in a stomacher (Lab
Blender 400, Seward Medical, London, UK). Serial dilutions were
made in 9 ml of one-quarter strength Ringer solution (Merck,
Darmstadt, Germany), and 0.1-ml portions were surface plated
onto duplicate PALCAM Listeria selective agar plates (Merck).
Plates were incubated at 30⬚C for 48 h and then examined for
typical L. monocytogenes colonies. The same procedure was re-
peated for the other sample.
When numbers of pathogens decreased below detection by
direct plating (⬍100 CFU g⫺1), the presence or absence of the
pathogen was determined by enrichment. A 25-g portion from
each package was aseptically transferred to separate stomacher
bags containing 225 ml of half Fraser Listeria selective enrich-
ment broth (Merck). After maceration, the samples were incubated
at 30⬚C for 24 h. Next, a 0.1-ml portion of the samples was trans-
ferred to 10 ml of Fraser Listeria selective enrichment broth
(Merck) and incubated at 35⬚C for 48 h before streak plating onto
PALCAM agar and incubating at 30⬚C for 24 h for characteristic
Listeria colonies.
Presumptive Listeria colonies were confirmed by biochemi-
cal tests. Suspect colonies were streaked onto Columbia blood
agar (Oxoid, Basingstoke, UK) and incubated at 37⬚C for 24 h.
Colonies that were beta-hemolytic were further tested for motility
at 25⬚C, catalase reaction, and carbohydrate acid production from
rhamnose (0.5%; Sigma, St. Louis, Mo.), xylose (0.5%; Sigma),
mannitol (0.5%; Sigma), and ␣-methyl-D-mannoside (0.5%; Sig-
ma) in purple carbohydrate broth base (Difco, Becton Dickinson,
Sparks, Md.).
Spoilage bacteria were enumerated by plating homogenate or
serial dilutions onto duplicate plates of the appropriate culture
medium. Aerobic mesophilic counts were obtained from plate
count agar (Merck) plates incubated at 25⬚C for 72 h; lactic acid
bacteria on deMan Rogosa Sharpe agar (Merck) overlaid with the
same medium and incubated at 25⬚C for 96 h under anaerobic
conditions; and micrococci and staphylococci on Staphylococcus
selective agar (Baird-Parker agar supplemented with Egg-Yolk
Tellurite Emulsion, Merck) incubated at 37⬚C for 48 h.
In addition to the microbiological analyses performed, the
pH of the samples was determined at each sampling interval with
a digital pH meter (Metrohm, 691, Herisau, Switzerland) with a
glass pH electrode.
J. Food Prot., Vol. 70, No. 10 SURVIVAL OF L. MONOCYTOGENES DURING THE STORAGE OF SLICED SALAMI
Data fitting. The data from plate counts (CFU per gram) of
duplicate samples from two independent experiments (n ⫽ 4)
were transformed to log values. To model the population of L.
monocytogenes as a function of time at each storage temperature,
the logistic Kamau equation (biphasic) (equation 1) (23) was used.
The selection of this model was based on the shape of survival
curves, which were linear or semisigmoidal (i.e., a death phase
followed by tailing):
log S ⫽ log
[ 2f
1 ⫹ exp(at)
⫹
2(1 ⫺ f )
1 ⫹ exp(bt) ] (1)
where f and (1 ⫺ f) represent the two inactivation phases of the
bacterial survival curve, a and b are the death rates for the two
phases of the bacterial survival curve, respectively, and t is the
time. The logistic Kamau equation was fitted to the logarithm of
the viable cell concentration by the in-house program DMFit 2
(Institute of Food Research, Reading, UK), which was kindly pro-
vided by Dr. J. Baranyi.
Two main parameters were estimated by the DMFit: a pri-
mary parameter, expressing the slope of the first inactivation phase
(a), and a second parameter, expressing the slope of the second
inactivation phase (b). Furthermore, by the fitted line, we esti-
mated the value of ta, representing the location of the joint of the
two inactivation phases (or the duration of the first phase). For an
accurate comparison between inactivation curves, the time needed
for 2- and 4-log reductions (t2D, t4D) in the L. monocytogenes
population was estimated. The calculations used are briefly ex-
plained below.
Da-values and Db-values were calculated by taking the neg-
ative reciprocal of the slope of the first and the second inactivation
phase, respectively. In all cases, the duration of first inactivation
phase was greater than the Da-value (ta ⬎ Da), thus indicating
that during ta, log reductions exceeded 1 Da. Therefore, the num-
ber of decimal reductions (NDa ) during the first inactivation phase
was estimated by the following formula (equation 2):
ta
NDa ⫽ (2)
Da
For a specific n-log reduction (nD), e.g., a 2- or 4-log reduction,
the ‘‘remaining D-value’’ (RD) was estimated by equation 3:
RD ⫽ nD ⫺ NDa (3)
When the RD was ⬍0, then the whole nD reduction had occurred
within the ta, and it was characterized by the Da; therefore, it was
calculated by formula 4:
tnD ⫽ ta ⫹ (RD ⫻ Db) (4)
When RD was ⬎0, a portion of the nD reduction had occurred
during the second phase of the inactivation curve, and it was char-
acterized by the Db; therefore, it was calculated by formula 5:
tnD ⫽ ta ⫹ (RD ⫻ Db) (5)
To determine differences between the initial populations (in-
oculum level) and to further enhance the direct comparison of the
inactivation curves, two more parameters were estimated: the time
needed for the L. monocytogenes population to reach the detection
limit (tdl) (2 log CFU/g) and the duration of extended survival
(ttail) (noncountable cells, but the presence of pathogen by enrich-
ment) before complete elimination of the pathogen (absence of
pathogen by enrichment).
To validate the effect of storage temperature, packaging, and
inoculum level on the survival of L. monocytogenes, individual
parameters (a, b, Nb, ta, t2D, t4D, tdl, and ttail) were used as de-
pendent variables for a mean comparison. The parameters were
2315
analyzed by analysis of variance by the GLM procedure of the
SPSS statistical package (SPSS 10.0.1 for Windows; SPSS, Inc.,
Chicago, Ill.). Tukey’s multiple-range test was used to compare
means. Means were considered significantly different at the P ⬍
0.05 level.
RESULTS AND DISCUSSION
The survival of L. monocytogenes following the post-
process contamination of sliced salami stored at 5, 15, and
25⬚C under air or vacuum was investigated. In two inde-
pendent sets of experiments, the surface of sliced salami
was inoculated with either a low (4.6 log CFU/g; batch A)
or a high (6.5 log CFU/g; batch B) population density of
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020
L. monocytogenes. Before inoculation, the absence of L.
monocytogenes was confirmed (with enrichment). The
shape of all inactivation curves was biphasic with an initial
rapid inactivation phase (described by the parameter a in
Table 1) within the first days of storage (indicated by pa-
rameter ta in Table 1), followed by a slower inactivation
phase (described by b in Table 1) or a profound ‘‘tailing’’
by the end of the storage period (Fig. 1). It should be noted,
however, that the absence of the pathogen was evident only
(confirmed by enrichment) at the end of the tested storage
period (i.e., 45 and 90 days for batches A and B, respec-
tively) under all conditions (Fig. 1). The duration of tailing
and the time for complete inactivation of L. monocytogenes
were quantified by the parameters ttail and tdl, respectively
(Table 2). Previous studies evaluating the effect of pro-
cessing and storage of fermented foods on the survival of
L. monocytogenes have also shown that inactivation of the
pathogen is often characterized by extended tailing (6, 33).
This phenomenon has been explained by the ability of bac-
terial cells to adapt to environmental stresses, such as acid,
heat, anaerobiosis, and nutrient starvation (26), through var-
ious cellular mechanisms (24, 45). In a recent study on the
acid tolerance of L. monocytogenes on frankfurters formu-
lated with antimicrobials and exposed periodically to sim-
ulated gastric fluid (pH 1.0), it was concluded that pathogen
resistance was dependent on the initial pathogen numbers
on the product as well as sufficient storage time for survi-
vors to develop acid resistance (37). Specifically, surviving
population of L. monocytogenes on frankfurters formulated
with 0.25% sodium diacetate and dipped in 2.5% lactic acid
(final pH, approximately 5.5) developed marked acid resis-
tance within 20 days of storage at 10⬚C (37). Furthermore,
it is known that bacterial cells become more resistant to
stress factors when they are attached to the surface of a
product compared with planktonically growing cells (22).
The parameters a, ta, b, t2D, t4D, tdl, and ttail were used to
quantify the effect of examined factors—storage tempera-
ture, packaging type, and inoculum level—on the biphasic
inactivation of L. monocytogenes. Specifically, a three-way
analysis of variance indicated that all the factors and their
interactions had significant (P ⬍ 0.05) effects on the in-
activation of L. monocytogenes.
With regard to storage temperature, our results clearly
demonstrated that the reduction of L. monocytogenes was
greater at room temperature (25⬚C) than at 15⬚C and chill
(5⬚C) temperature (Fig. 1). This effect was more evident
2316 GOUNADAKI ET AL. J. Food Prot., Vol. 70, No. 10

TABLE 1. Inactivation kinetics of Listeria monocytogenes during storage of sliced salamia

Temp (⬚C) Inoculum Package type a (day⫺1)b ta (day)c b (day⫺1)d Nbe R2f

5 Low Air ⫺0.20 ABC (0.01)g 08.86 BC (0.79) ⫺0.03 CD (0.00) 2.95 BC (0.05) 0.86, 0.91
Vacuum ⫺0.35 D (0.02) 03.67 A (0.18) ⫺0.04 BC (0.00) 3.32 C (0.09) 0.81, 0.83
High Air ⫺0.17 AB (0.01) 10.51 BCD (2.50) ⫺0.06 A (0.00) 4.55 D (0.25) 0.91, 0.93
Vacuum ⫺0.09 A (0.00) 37.70 F (0.00) ⫺0.06 A (0.00) 2.24 A (0.00) 0.94, 0.94
15 Low Air ⫺0.19 AB (0.00) 13.00 CD (0.37) ⫺0.00 F (0.00) 2.00 A (0.00) 0.95, 0.97
Vacuum ⫺0.24 BCD (0.10) 06.28 AB (2.76) ⫺0.04 BC (0.01) 3.18 C (0.29) 0.96, 0.99
High Air ⫺0.28 BCD (0.04) 14.71 D (2.53) ⫺0.02 E (0.01) 2.50 AB (0.27) 0.94, 0.96
Vacuum ⫺0.19 AB (0.00) 20.49 E (0.69) ⫺0.02 DE (0.01) 2.94 BC (0.06) 0.95, 0.97
25 Low Air ⫺0.30 BCD (0.04) 07.62 AB (0.32) ⫺0.01 EF (0.00) 2.12 A (0.01) 0.90, 0.92
Vacuum ⫺0.38 D (0.07) 03.72 A (0.48) ⫺0.04 B (0.00) 3.28 C (0.06) 0.90, 0.98
High Air ⫺0.33 CD (0.00) 14.59 D (0.04) ⫺0.00 F (0.00) 2.03 A (0.02) 0.96, 0.97

Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020

Vacuum ⫺0.20 ABC (0.00) 22.48 E (0.45) ⫺0.00 F (0.00) 2.00 A (0.00) 0.98, 0.99
a Values in parentheses are ⫾ standard deviation.
b Death rate of the first phase of inactivation curve (linear part).
c Duration (in days) of the first phase of the inactivation curve.
d Death rate of the second phase of the inactivation curve (tail).
e Cell fraction (log CFU per gram) at the time the second phase of the inactivation curve starts.
f Adjusted R2 statistics of the fitting (minimum, maximum).
g Means within a column sharing at least a common letter are not significantly different at the P ⬍ 0.05 level.

during the second phase of the inactivation curve (F ⫽
195.84, P ⬍ 0.05). The results suggest that storage of sa-
lami above refrigeration enhances destruction of L. mono-
cytogenes. Previous studies have also shown that storage
temperature is among the most important factors that can
influence the survival of foodborne pathogens in low pH
foods, such as apple cider, mayonnaise, eggplant salad, and
fermented sausages (9, 10, 17, 20, 36, 41). More specifi-
cally, studies on the viability of E. coli O157:H7 during the
manufacture and storage of either soudjouk sticks (pH 4.48
to 5.40) (5) or salami (pH 4.82) (15) have shown that num-
bers of E. coli O157:H7 decreased faster during storage at

FIGURE 1. Survival of Listeria monocytogenes (n ⫽ 4) during the storage of sliced salami at 5⬚C (䡵), 15⬚C (䉱), and 25⬚C (䢇). (A)
Samples with low inoculum (batch A). (B) Samples with high inoculum (batch B). Filled symbols, air-packaged samples (1); open
symbols, vacuum-packaged samples (2). Dashed line: counts were below the detection limit (horizontal dashed line) by direct plating,
and the presence or absence of the pathogen was determined by enrichment.
J. Food Prot., Vol. 70, No. 10 SURVIVAL OF L. MONOCYTOGENES DURING THE STORAGE OF SLICED SALAMI 2317

TABLE 2. Duration of Listeria monocytogenes reduction in sliced fermented sausages packed under air or vacuum at three different
temperaturesa
Temp (⬚C) Inoculum Package type t 2Db t 4Dc t dld t taile

5 Low Air 16.42 BCD (2.86)f NDg 16.42 ABC (2.86) 28.58 AB (2.86)
Vacuum 23.31 D (4.36) ND 23.31 CD (4.36) 21.69 A (4.36)
High Air 13.26 ABC (1.75) 44.90 D (0.38) 44.90 E (0.38) 45.10 D (0.38)
Vacuum 22.48 D (0.11) 44.97 D (0.21) 44.97 E (0.21) 45.03 D (0.21)
15 Low Air 10.51 ABC (0.14) ND 10.51 AB (0.14) 34.49 BC (0.14)
Vacuum 23.91 D (4.56) ND 23.91 CD (4.56) 21.09 A (4.56)
High Air 07.30 A (0.94) 14.59 AB (1.88) 14.59 ABC (1.88) 75.41 F (1.88)
Vacuum 10.73 ABC (0.11) 29.48 C (6.15) 29.48 D (6.15) 60.52 E (6.15)
25 Low Air 06.79 A (0.81) ND 06.79 A (0.81) 38.21 CD (0.81)
Vacuum 18.04 CD (2.64) ND 18.04 BC (2.64) 26.96 AB (2.64)

Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020

High Air 05.98 A (0.03) 11.96 A (0.06) 11.96 AB (0.06) 78.04 F (0.06)
Vacuum 09.79 AB (0.02) 19.59 B (0.04) 19.59 BC (0.04) 70.41 F (0.04)
a Values are in days ⫾ standard deviation; n ⫽ 4.
b Time (days) needed for a 2-log reduction.
c Time (days) needed for a 4-log reduction.
d Time (days) needed for a population to reach detection limit (2 log CFU/g).
e Duration (days) of extended survival (noncountable cells but presence of pathogen by enrichment) before complete elimination (absence

of pathogen by enrichment).
f Means within a column sharing at least a common letter are not significantly different at the P ⬍ 0.05 level.
g ND, not determined.

21⬚C than at 4⬚C. Furthermore, Chikthimmah and Knabel
(10) showed enhanced survival of L. monocytogenes and
E. coli O157:H7 during storage of Lebanon bologna slices
(pH 4.6) at 3.6⬚C compared with storage at 13⬚C. Although
indicative of the potential temperature effects on the fate of
L. monocytogenes, results of the above-mentioned studies
are not comparable because of the large diversity in process
parameters and product characteristics (e.g., pH, water ac-
tivity, variations in time and temperature).
The type of packaging in combination with storage
temperature affected (P ⬍ 0.05) the duration of the first
inactivation phase (ta), as well as the destruction rate (b) of
the second inactivation phase (Table 1). More specifically,
vacuum packaging resulted in the slower destruction of L.
monocytogenes than aerobic packaging, and this effect in-
creased as storage temperature decreased (Fig. 1). Our re-
sults are consistent with results of previous studies that
demonstrated that the storage of salami (15) or pepperoni
slices (16) at ambient temperature (21⬚C) or aerobic con-
ditions was more effective at reducing levels of E. coli
O157:H7 than refrigeration (4⬚C) or storage under vacuum.
Similarly, it has been reported that aerobic conditions re-
sulted in higher destruction of L. monocytogenes in simu-
lated salami matrix than under vacuum at 20⬚C (42). How-
ever, in the same study, storage at 4⬚C under vacuum re-
sulted in slightly higher destruction of L. monocytogenes
than in aerobic storage (42). In general, storage recommen-
dations for salami suggest refrigeration for packaged prod-
ucts for an indefinite period and for only 3 weeks, once the
products are opened (39). The present study indicates that
vacuum packaging enhances survival of L. monocytogenes
compared with aerobic storage (unpackaged products), es-
pecially at low temperatures. Therefore, our results may
contribute to the improvement of guidelines for the storage
of packaged or unpackaged fermented salami.
The initial inoculation level also appeared to affect (P
⬍ 0.05) the duration and rate of the first inactivation phase
of L. monocytogenes (Table 1). More specifically, high in-
oculum (6.5 log CFU/g) resulted in a shorter t2D (faster
destruction) than low inoculum (4.6 log CFU/g; Table 2).
In addition, although numbers of L. monocytogenes were
reduced below the detection limit in all cases, the time
needed for a reduction to the detection limit (tdl: F ⫽ 91.03,
P ⬍ 0.05) and complete elimination (ttail: F ⫽ 852.24, P
⬍ 0.05) was longer for the high inoculum (Table 2). Similar
findings have been reported by Yoon et al. (44), who ob-
served that high levels (6.8 log CFU/cm2) of E. coli O157:
H7 experienced faster inactivation than low levels (3.8 log
CFU/cm2) during drying of beef jerky that had been sub-
jected acidic marinade (pH on meat surface, 4.23) before
drying. It has also been reported that stationary-phase
gram-negative cells of low density are more sensitive to
low pH than those of high cell density (12). On the other
hand, Buchanan et al. (4) observed that inactivation rates
of L. monocytogenes in liquid media were independent of
initial population density. However, the differences between
our findings and those of Buchanan et al. are likely asso-
ciated with differences in the number and type of L. mon-
ocytogenes strains as well as in the type of media (liquid
media versus food matrix). More specifically, in the study
of Buchanan et al., a mixture of three strains of L. mono-
cytogenes (Scott A, HO-VJ-S, and V-7) was inoculated in
liquid media, whereas we used a single L. monocytogenes
Scott A strain on the surface of salami slices. Thus, the
results of Buchanan et al. likely represent the survival be-
havior of the sturdiest (most resistant) strain, and such a
2318 GOUNADAKI ET AL. J. Food Prot., Vol. 70, No. 10

strain (i.e., showing the highest counts) may vary depend-

AB
A

A
B
ing on the experimental conditions. Thus, different counts

(⫾0.00)
(⫾0.09)
(⫾0.00)

(⫾0.01)
of L. monocytogenes between treatments may eventually

25⬚C
represent different strains. Moreover, data from broth and
food studies are not directly comparable, because factors

4.11
4.30
⬍2.00
4.51
that could potentially affect microbial survival in foods,
such as food structure and microbial interactions, are com-
monly ignored in laboratory media. For example, it has
been reported that the natural microbial flora of a food en-
Vacuum package

D
D
B
B
(⫾0.02) vironment may protect or sensitize pathogens to acid stress
(⫾0.03)
(⫾0.08)
(⫾0.00)
(34). Other researchers have attributed the inhibition of L.
15⬚C

monocytogenes during storage of fermented foods to the

5.74
5.54
4.02
4.57 competition of this pathogen with high populations of lac-

Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020

tobacilli (3, 21). The extent of inhibition is closely related
to the high numbers of lactic acid bacteria present (high
ratio of lactobacilli:pathogen) (1), as also evident in our
AB

study (Table 3).

A
F
F
(⫾0.18)
(⫾0.03)
(⫾0.08)
(⫾0.02)

Overall, the findings of the present study suggest that

Day 45 of batch A stored under:
TABLE 3. Changes in the initial and final microbial association and in the pH of sliced salami, of uninoculated (control) samplesa

5⬚C

the established recommendations for refrigerated storage of

packaged (vacuum) salami enhance the survival of L. mono-
6.59
6.69
4.02
4.51

cytogenes. Furthermore, the observed slow destruction of

L. monocytogenes at lower temperatures indicates that in a
postprocess contamination event, sliced salami is a source
of exposure of consumers to L. monocytogenes. Therefore,
A
A
A
C

(⫾0.00)
(⫾0.02)
(⫾0.07)

(⫾0.00)

our findings may also contribute to the more reliable as-

sessment of L. monocytogenes exposure, depending on the
25⬚C

c Means within a row sharing at least a common letter are not significantly different at the P ⬍ 0.05 level.

storage conditions (temperature and packaging) at home.

5.45
3.29
⬍2.00
4.46

However, the significance of exposure remains to be eval-

uated, for until now, contamination levels lower than 103
CFU/g have not, to our knowledge, been documented for
foods implicated in cases of invasive listeriosis (40). More-
over, further research is needed to quantify the survival po-
A
A
B
C
Air package

(⫾0.00)
(⫾0.01)
(⫾0.63)
(⫾0.02)

tential of various single or multiple L. monocytogenes

15⬚C

strains at different inoculation levels in traditional sausages

of different geographic origins and manufacturing practices.
Each value is the mean (⫾ standard deviation) of duplicate product samples.
4.90
4.72
2.45
4.47

ACKNOWLEDGMENTS
This study has been partly carried out with the financial support of
the Commission of the European Communities, specific Research and
A
A
E
E

Technological Development program Quality of Life and Management of

(⫾0.09)
(⫾0.08)
(⫾0.23)
(⫾0.05)

Living Resources, Key Action 1—Health Food and Environment, project

QLK1-CT-2002-02240 (TRADISAUSAGE). It does not necessarily reflect
5⬚C

the Commission’s views and in no way anticipates its future policy in this
6.21
6.02
3.01
4.48

area.

REFERENCES
1. Adams, M. R., and L. Nicolaides. 1997. Review of the sensitivity
(⫾0.02)b Gc

(⫾0.05) AB
(⫾0.04) AB

Values are in log CFU per gram; n ⫽ 4.

of different foodborne pathogens to fermentation. Food Control 8:

(⫾0.05) G

227–239.
Day 0

2. Angelidis, A. P., and K. Koutsoumanis. 2006. Prevalence and con-

centration of Listeria monocytogenes in sliced ready-to-eat meat
products in the Hellenic retail market. J. Food Prot. 69:938–942.
7.45
7.78
4.47
4.47

3. Brashears, M. M., and W. A. Durre. 1999. Antagonistic action of

Lactobacillus lactis towards Salmonella spp. and Escherichia coli
O157:H7 during growth and refrigerated storage. J. Food Prot. 62:
1336–1340.
Staphylococcus spp.
Lactic acid bacteria
Total viable counts

4. Buchanan, R. L., M. H. Golden, R. C. Whiting, J. G. Philips, and J.

L. Smith. 1994. Non-thermal inactivation models for Listeria mono-
cytogenes. J. Food Sci. 59:179–188.
5. Calicioglu, M., N. G. Faith, D. R. Buege, and J. N. Luchansky. 2001.
Validation of a manufacturing process for fermented, semidry Turk-
ish soudjouk to control Escherichia coli O157:H7. J. Food Prot. 64:
pH

1156–1161.
a
b
J. Food Prot., Vol. 70, No. 10 SURVIVAL OF L. MONOCYTOGENES DURING THE STORAGE OF SLICED SALAMI
6. Calicioglu, M., J. N. Sofos, J. Samelis, P. A. Kendall, and G. C.
Smith. 2002. Destruction of acid- and non-adapted Listeria mono-
cytogenes during drying and storage of beef jerky. Food Microbiol.
19:545–559.
7. Centers for Disease Control and Prevention. 2000. Multistate out-
break of listeriosis—United States. Morb. Mortal. Wkly. Rep. 49:
1129–1130.
8. Centers for Disease Control and Prevention. 2002. Public health dis-
patch: outbreak of listeriosis—northeastern United States. Morb.
Mortal. Wkly. Rep. 51:950–951.
9. Chikthimmah, N., R. B. Guyer, and S. J. Knabel. 2001. Validation
of a 5-log reduction of Listeria monocytogenes following simulated
commercial processing of Lebanon bologna in a model system. J.
Food Prot. 64:873–876.
10. Chikthimmah, N., and S. J. Knabel. 2001. Survival of Escherichia
coli O157:H7, Salmonella Typhimurium, and Listeria monocyto-
genes in and on vacuum packaged Lebanon bologna stored at 3.6
and 13.0⬚C. J. Food Prot. 64:958–963.
11. Commission of the European Communities. 2005. Commission
regulation (EC) 2073/2005 of 15 November 2005 on microbio-
logical criteria for foodstuffs. Off. J. Eur. Union L338:1–26. Avail-
able at: http://eur-lex.europa.eu/LexUriServ/site/en/oj/2005/l㛮338/l㛮
33820051222en00010026.pdf. Accessed 20 February 2006.
12. Datta, A. R., and M. M. Benjamin. 1999. Cell density dependent
acid sensitivity in stationary phase cultures of enterohemorrhagic
Escherichia coli O157:H7. FEMS Microbiol. Lett. 181:289–295.
13. Drosinos, E. H., M. Mataragas, S. Veskovic-Moracanin, J. Gasparik-
Reichardt, M. Hadziosmanovic, and D. Alagic. 2006. Quantifying
nonthermal inactivation of Listeria monocytogenes in European fer-
mented sausages using bacteriocinogenic lactic acid bacteria or their
bacteriocins: a case study for risk assessment. J. Food Prot. 69:
2648–2663.
14. European Commission Research Proposal QLK1 CT-2002-02240.
Assessment and improvement of safety of traditional dry sausages
from producers to consumers (TRADISAUSAGE). Available at:
http://www2.clermont.inra.fr/tradisausage/index.htm. Accessed 30
May 2006.
15. Faith, N. G., N. Parniere, T. Larson, T. D. Lorang, C. W. Kaspar,
and J. B. Luchansky. 1998. Viability of Escherichia coli O157:H7
in salami following conditioning of batter, fermentation and drying
of sticks, and storage of slices. J. Food Prot. 61:377–382.
16. Faith, N. G., R. K. Wierzba, A. M. Ihnot, A. R. Roering, T. D.
Lorang, C. W. Kaspar, and J. B. Luchansky. 1998. Survival of Esch-
erichia coli O157:H7 in full- and reduced-fat pepperoni after man-
ufacture of sticks, storage of slices at 4⬚C or 21⬚C under air and
vacuum, and baking of slices on frozen pizza at 135, 191 and 246⬚C.
J. Food Prot. 61:383–389.
17. Fisher, T. L., and D. A. Golden. 1998. Fate of Escherichia coli O157:
H7 in ground apples used in cider production. J. Food Prot. 61:
1372–1374.
18. Giovannacci, I., C. Ragimbeau, S. Queguiner, G. Salvat, J. L. Ven-
deuvre, V. Carlier, and G. Ermel. 1999. Listeria monocytogenes in
pork slaughtering and cutting plants use of RAPD, PFGE and PCR–
REA for tracing and molecular epidemiology. Int. J. Food Microbiol.
53:127–140.
19. Glass, K. A., and M. P. Doyle. 1999. Fate of Listeria monocytogenes
in processed meat products during refrigerated storage. Appl. Envi-
ron. Microbiol. 55:1565–1569.
20. Hew, C. M., M. N. Hajmeer, T. B. Farver, J. M. Glover, and D. O.
Cliver. 2005. Survival of Listeria monocytogenes in experimental
chorizos. J. Food Prot. 68:324–330.
21. Hugas, M. M., M. Garriga, M. T. Aymerich, and J. M. Monfort.
1995. Inhibition of Listeria in dry fermented sausages by the bac-
teriocinogenic Lactobacillus sake CTC494. J. Appl. Microbiol. 79:
322–330.
22. Humphrey, T. J., S. J. Wilde, and R. J. Rowbury. 1997. Heat toler-
ance of Salmonella typhimurium DT104 isolates attached to muscle
tissue. Lett. Appl. Microbiol. 25:265–268.
23. Kamau, D. N., S. Doores, and K. M. Prutt. 1990. Enhanced thermal
destruction of Listeria monocytogenes and Staphylococcus aureus by
2319
the lactoperoxidase system. Appl. Environ. Microbiol. 56:2711–
2716.
24. Ko, R., and L. T. Smith. 1999. Identification of an ATP-driven, os-
moregulated glycine betaine transport system in Listeria monocyto-
genes. Appl. Environ. Microbiol. 65:4040–4048.
25. Kyungwha, L., and M. Azlin. 2007. Inhibition of Escherichia coli
O157:H7, Listeria monocytogenes and Staphylococcus aureus on
sliced roast beef by cetylpyridinium chloride and acidified sodium
chlorite. Food Microbiol. 24:89–94.
26. Leistner, L. 2000. Basic aspects of food preservation by hurdle tech-
nology. Int. J. Food Microbiol. 55:181–186.
27. Levine, P., B. Rose, S. Green, G. Ransom, and W. Hill. 2001. Path-
ogen testing of ready-to-eat meat and poultry products collected at
federally inspected establishments in the United States, 1990 to
1999. J. Food Prot. 64:1188–1193.
28. Lianou, A., J. D. Stopforth, Y. Yoon, M. Wiedmann, and J. N. Sofos.
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020
2006. Growth and stress resistance variation in culture broth among
Listeria monocytogenes strains of various serotypes and origins. J.
Food Prot. 69:2640–2647.
29. Moore, J. E. 2004. Gastrointestinal outbreaks associated with fer-
mented meats. Meat Sci. 67:565–568.
30. Morales, P., J. Calzada, and M. Nunez. 2006. Effect of high-pressure
treatment on the survival of Listeria monocytogenes Scott A in sliced
vacuum-packaged Iberian and Serrano cured hams. J. Food Prot. 69:
2539–2543.
31. Nesbakken, T., G. Kapperud, and D. A. Caugant. 1996. Pathways of
Listeria monocytogenes contamination in the meat processing indus-
try. Int. J. Food Microbiol. 31:161–171.
32. Nightingale, K. K., H. Thippareddi, R. K. Phebus, J. L. Marsden,
and A. L. Nutsch. 2006. Validation of a traditional Italian-style sa-
lami manufacturing process for control of Salmonella and Listeria
monocytogenes. J. Food Prot. 69:794–800.
33. Samelis, J., A. Kakouri, I. N. Savvaidis, K. Riganakos, and M. G.
Kontominas. 2005. Use of ionizing radiation doses of 2 and 4 kGy
to control Listeria spp. and Escherichia coli O157:H7 on frozen meat
trimmings used for dry fermented sausage production. Meat Sci. 70:
189–195.
34. Samelis, J., J. N. Sofos, P. A. Kendall, and G. C. Smith. 2001. In-
fluence of the natural microbial flora on the acid tolerance response
of Listeria monocytogenes in a model system of fresh meat decon-
tamination fluids. Appl. Environ. Microbiol. 67:2410–2420.
35. Schultze, K. K., R. H. Linton, M. A. Cousin, J. B. Luchansky, and
M. L. Tamplin. 2006. A predictive model to describe the effects of
temperature, sodium lactate, and sodium diacetate on the inactivation
of a serotype 4b strain of Listeria monocytogenes in a frankfurter
slurry. J. Food Prot. 69:1552–1560.
36. Skandamis, N. P., and G. J. E. Nychas. 2000. Ecophysiological at-
tributes of Salmonella typhimurium in liquid culture and within gel-
atin gel with or without the addition of oregano essential oil. World
J. Microbiol. Biotechnol. 16:31–35.
37. Stopforth, J. D., Y. Yoon, I. M. Barmpalia, J. Samelis, P. N. Skan-
damis, and J. N. Sofos. 2005. Reduction of Listeria monocytogenes
populations during exposure to a stimulated gastric fluid following
storage of inoculated frankfurters formulated and treated with pre-
servatives. Int. J. Food Microbiol. 99:309–319.
38. Thevenot, D., M. L. Delignette-Muller, S. Christieans, and C. Ver-
nozy-Rozand. 2005. Fate of Listeria monocytogenes in experimen-
tally contaminated French sausages. Int. J. Food Microbiol. 101:
189–200.
39. U.S. Department of Agriculture. 2003. Focus on: sausages. Food Safe-
ty and Inspection Service, Washington, D.C. Available at: http://
www.fsis.usda.gov/fact㛮sheets/Sausage㛮and㛮Food㛮Safety/index.asp.
Accessed 30 March 2003.
40. U.S. Food and Drug Administration, Center for Food Safety and
Applied Nutrition (FDA/CFSAN). 2003. Quantitative assessment of
the relative risk to public health from foodborne Listeria monocy-
togenes among selected food categories of ready-to-eat foods. Avail-
able at: http://www.fsis.usda.gov/Science/Risk㛮Assessments/index.
asp. Accessed 20 August 2004.
41. Weagent, S. D., J. L. Bryant, and D. H. Bark. 1994. Survival of
2320 GOUNADAKI ET AL.
Escherichia coli O157:H7 in mayonnaise and mayonnaise-based
sauces at room and refrigerated temperatures. J. Food Prot. 57:629–
631.
42. Williams, R. C., and D. A. Golden. 2001. Influence of modified
atmospheric storage, lactic acid, and NaCl on survival of sublethally
heat-injured Listeria monocytogenes. Int. J. Food Microbiol. 64:
379–386.
43. Yoon, K. S., C. N. Burnette, K. A. Abou-Zeid, and R. C. Whitting.
2004. Control of growth and survival of Listeria monocytogenes on
smoked salmon by combined potassium lactate and sodium diacetate
J. Food Prot., Vol. 70, No. 10
and freezing stress during refrigeration and frozen storage. J. Food
Prot. 67:2465–2471.
44. Yoon, Y., M. Calicioglu, P. A. Kendall, G. C. Smith, and J. N. Sofos.
2005. Influence of inoculum level and acidic marination on inacti-
vation of Escherichia coli O157:H7 during drying and storage of
beef jerky. Food Microbiol. 22:423–431.
45. Yoon, Y., P. N. Skandamis, P. A. Kendall, G. C. Smith, and J. N.
Sofos. 2006. A predictive model for the effect of temperature and
predrying treatments in reducing Listeria monocytogenes populations
during drying of beef jerky. J. Food Prot. 69:62–70.
Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/10/2313/1677197/0362-028x-70_10_2313.pdf by guest on 13 October 2020