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

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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- foodborne pathogens such as L. monocytogenes may over-
ganism that is ubiquitous in nature and common in foods come these hurdles in French sausages (38), chorizo sau-
of both plant and animal origin. It is recognized as a po- sages (20), and Italian-style salami (32). Although L. mono-
tential contaminant of ready-to-eat (RTE) products, as sev- cytogenes is among the most frequently detected pathogens
eral outbreaks of L. monocytogenes were found to be as- in fermented sausages, reaching prevalence levels of up to
sociated with the consumption of refrigerated RTE products 30% (2, 27), clinical listeriosis cases have yet to be linked
(7, 8). The high mortality rate (30%) associated with lis- to the consumption of fermented sausages contaminated
teriosis has caused L. monocytogenes to be considered a with L. monocytogenes (32). So far, there is only one epi-
public health hazard (40). However, the high incidence of demiologic association of L. monocytogenes with salami in
this microorganism (18) and its ability to persist within the Philadelphia, but no confirmed outbreak due to the con-
food-processing environment for years (31) make control sumption of such products with Listeria has, to our knowl-
of L. monocytogenes a challenge for the food industry. edge, been reported (29). However, salami belongs to the
Fermented sausages such as salami are widely popular category of commercially available RTE meat products for
deli meat products all over the world. Their sales have sig-
which the U.S. Food and Drug Administration–Center for
nificantly increased in the last two decades on both sides
Food Safety and Applied Nutrition (40) has clearly outlined
of the Atlantic Ocean. They are consumed as appetizers
the need for further research related to survival and growth
(RTE), side dishes, dips, or sandwich fillers, and they are
kinetic data of L. monocytogenes.
purchased as packed retail products or per weight through
The intrinsic characteristics (e.g., low pH, water activ-
the deli section of supermarkets, delicatessen stores, or food
service operations. They are considered one of the safest ity) of fermented sausages, as well as the refrigeration tem-
food groups for humans because their manufacture involves peratures, have been employed as fundamental strategies
a combination of hurdles (low pH, low water activity, pres- for their preservation (39). In addition, dry fermented sau-
ence of competitive microflora, and curing salts) to patho- sages of water activity ⬍0.90 and pH of approximately 4.5
gen survival or growth. However, studies have shown that (14) may be stored at ambient temperatures. Indeed, prelim-
inary studies in our laboratory regarding the preferences
* Author for correspondence. Tel: ⫹302105294684; Fax: ⫹302105294683; and handling habits of European Union consumers on fer-
E-mail: [email protected]. mented sausages show that 40.2% of consumers store these
2314 GOUNADAKI ET AL. J. Food Prot., Vol. 70, No. 10

products at room temperature (25⬚C in kitchen or 15⬚C in slices (50 g) were placed into clear polyethylene bags, packaged
cellar) (14). However, the risk associated with the survival under air or vacuum, and stored at 5, 15, or 25⬚C. Samples were
of L. monocytogenes is likely to differ among fermented stored for 45 or 90 days for low or high inoculum level, respec-
sausages, mainly because of the complexity and variety of tively.
Vacuum packages were performed with a Henkovac 1900
manufacturing recipes used. Thus, there is a need for sys-
(Hertogenbosch, The Netherlands). For samples stored under air,
tematic scientific knowledge about the safety of such foods.
the Ziploc-type bags were fastened about 0.5 cm from the top to
For marketing and product quality reasons, nowadays achieve an unoccupied volume of 650 ml. Two different batches
fermented sausages are usually vacuum packaged and of salami, within a 3-month period (batch A and batch B), were
stored, distributed, and displayed at refrigeration tempera- prepared under the same manufacturing conditions. Both batches
ture (40), conditions that positively affect their appearance were individually inoculated as described above, with different
and shelf life. According to a preliminary survey on con- inoculum levels, which resulted in a low (batch A, 4.6 log CFU/g)
sumer preferences for dry fermented sausages, the majority and a high (batch B, 6.5 log CFU/g) population density of L.
of consumers prefer to buy these RTE products as whole monocytogenes. The above procedures were performed twice, and

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sausages, while an increasing percentage of consumers pur- the samples were analyzed in duplicate.
chase them sliced and vacuum packaged either from the
Microbiological analysis. At specific time intervals (0, 3, 6,
processing plants or at retail points at the time of sale (14).
10, 13, 17, 20, 24, 27, 31, 38, and 45 days of storage for batch
Before consumption, the majority of the consumers (81.1%) A and 0, 3, 6, 10, 13, 17, 20, 24, 27, 31, 38, 45, 58, and 90 days
repack these products in plastic (bags) (14). Although there of storage for batch B), two packages were removed from storage
is excessive documentation on the fate of L. monocytogenes and analyzed independently. Specifically, from each package, 25-g
on vacuum-packaged fermented sausages (9, 10, 19), there portions of salami were aseptically transferred to a stomacher bag
is limited information on the comparative evaluation of the containing 225 ml of sterile 0.1% peptone water with salt (NaCl,
survival of pathogens under air or vacuum. Thus, there is 0.85%, wt/vol) and macerated for 1 min in a stomacher (Lab
a need for data related to the behavior of L. monocytogenes Blender 400, Seward Medical, London, UK). Serial dilutions were
in deli meat products packaged under vacuum or unpacked. made in 9 ml of one-quarter strength Ringer solution (Merck,
Such data are also of value for the implementation of mi- Darmstadt, Germany), and 0.1-ml portions were surface plated
crobiological criteria for foods illustrated by European onto duplicate PALCAM Listeria selective agar plates (Merck).
Plates were incubated at 30⬚C for 48 h and then examined for
Union Regulation 2073/2005 (11). Therefore, the objective
typical L. monocytogenes colonies. The same procedure was re-
of the present study was to evaluate the effect of packaging
peated for the other sample.
under air or vacuum on the survival of postprocessing L. When numbers of pathogens decreased below detection by
monocytogenes contamination on slices of dry fermented direct plating (⬍100 CFU g⫺1), the presence or absence of the
salami stored at 5, 15, and 25⬚C. pathogen was determined by enrichment. A 25-g portion from
each package was aseptically transferred to separate stomacher
MATERIALS AND METHODS bags containing 225 ml of half Fraser Listeria selective enrich-
Preparation of bacterial inoculum. L. monocytogenes Scott ment broth (Merck). After maceration, the samples were incubated
A was used for the inoculation of sliced salami (kindly provided at 30⬚C for 24 h. Next, a 0.1-ml portion of the samples was trans-
by Dr. Eddy Smid, Agricultural Research Institute, Wageningen, ferred to 10 ml of Fraser Listeria selective enrichment broth
The Netherlands). This strain was selected because it is often used (Merck) and incubated at 35⬚C for 48 h before streak plating onto
in low temperature or acid challenge studies because of its clinical PALCAM agar and incubating at 30⬚C for 24 h for characteristic
origin and the strong epidemiologic association of serotype 4b Listeria colonies.
with human listeriosis (28). Furthermore, the use of individual L. Presumptive Listeria colonies were confirmed by biochemi-
monocytogenes strains of various origin, as applied by several cal tests. Suspect colonies were streaked onto Columbia blood
researchers (13, 25, 30, 35, 43), may provide representative data agar (Oxoid, Basingstoke, UK) and incubated at 37⬚C for 24 h.
equal to those of studies with multiple clinical or outbreak iso- Colonies that were beta-hemolytic were further tested for motility
lates. The stock culture was activated from frozen beads (Protect, at 25⬚C, catalase reaction, and carbohydrate acid production from
Technical Service Consultants Ltd., Lancashire, UK) in 10 ml of rhamnose (0.5%; Sigma, St. Louis, Mo.), xylose (0.5%; Sigma),
tryptone soy broth (TSB; LAB M, International Diagnostics mannitol (0.5%; Sigma), and ␣-methyl-D-mannoside (0.5%; Sig-
Group Plc., Bury, UK) for 18 h at 30⬚C. A 0.1-ml portion of the ma) in purple carbohydrate broth base (Difco, Becton Dickinson,
activated culture was transferred to 100 ml of TSB and incubated Sparks, Md.).
at 30⬚C for 18 h. Bacteria were harvested after centrifugation at Spoilage bacteria were enumerated by plating homogenate or
4,000 ⫻ g for 10 min at 4⬚C. The cell pellet was washed and serial dilutions onto duplicate plates of the appropriate culture
resuspended twice in 100 ml of 0.85% saline before inoculation. medium. Aerobic mesophilic counts were obtained from plate
count agar (Merck) plates incubated at 25⬚C for 72 h; lactic acid
Preparation of salami. Sliced salami used in this study was bacteria on deMan Rogosa Sharpe agar (Merck) overlaid with the
obtained from a commercial sausage manufacturing company. The same medium and incubated at 25⬚C for 96 h under anaerobic
salami was processed on a meat slicer preset to yield 6- to 7-g conditions; and micrococci and staphylococci on Staphylococcus
slices and transferred to the laboratory for surface inoculation. The selective agar (Baird-Parker agar supplemented with Egg-Yolk
upper surface of each slice was inoculated with 0.025 ml of the Tellurite Emulsion, Merck) incubated at 37⬚C for 48 h.
L. monocytogenes culture by spreading the inoculum over the en- In addition to the microbiological analyses performed, the
tire surface with a sterile bent glass rod. Following a 10-min rest- pH of the samples was determined at each sampling interval with
ing period for bacterial attachment, the slices were flipped over, a digital pH meter (Metrohm, 691, Herisau, Switzerland) with a
and the other side was inoculated. Six to eight of the inoculated glass pH electrode.
J. Food Prot., Vol. 70, No. 10 SURVIVAL OF L. MONOCYTOGENES DURING THE STORAGE OF SLICED SALAMI 2315

Data fitting. The data from plate counts (CFU per gram) of analyzed by analysis of variance by the GLM procedure of the
duplicate samples from two independent experiments (n ⫽ 4) SPSS statistical package (SPSS 10.0.1 for Windows; SPSS, Inc.,
were transformed to log values. To model the population of L. Chicago, Ill.). Tukey’s multiple-range test was used to compare
monocytogenes as a function of time at each storage temperature, means. Means were considered significantly different at the P ⬍
the logistic Kamau equation (biphasic) (equation 1) (23) was used. 0.05 level.
The selection of this model was based on the shape of survival
curves, which were linear or semisigmoidal (i.e., a death phase RESULTS AND DISCUSSION
followed by tailing):
The survival of L. monocytogenes following the post-
log S ⫽ log
[ 2f
1 ⫹ exp(at)
⫹
2(1 ⫺ f )
1 ⫹ exp(bt) ] (1)
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
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 was inoculated with either a low (4.6 log CFU/g; batch A)
phases of the bacterial survival curve, respectively, and t is the or a high (6.5 log CFU/g; batch B) population density of

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time. The logistic Kamau equation was fitted to the logarithm of L. monocytogenes. Before inoculation, the absence of L.
the viable cell concentration by the in-house program DMFit 2 monocytogenes was confirmed (with enrichment). The
(Institute of Food Research, Reading, UK), which was kindly pro- shape of all inactivation curves was biphasic with an initial
vided by Dr. J. Baranyi. rapid inactivation phase (described by the parameter a in
Two main parameters were estimated by the DMFit: a pri- Table 1) within the first days of storage (indicated by pa-
mary parameter, expressing the slope of the first inactivation phase rameter ta in Table 1), followed by a slower inactivation
(a), and a second parameter, expressing the slope of the second
phase (described by b in Table 1) or a profound ‘‘tailing’’
inactivation phase (b). Furthermore, by the fitted line, we esti-
mated the value of ta, representing the location of the joint of the
by the end of the storage period (Fig. 1). It should be noted,
two inactivation phases (or the duration of the first phase). For an however, that the absence of the pathogen was evident only
accurate comparison between inactivation curves, the time needed (confirmed by enrichment) at the end of the tested storage
for 2- and 4-log reductions (t2D, t4D) in the L. monocytogenes period (i.e., 45 and 90 days for batches A and B, respec-
population was estimated. The calculations used are briefly ex- tively) under all conditions (Fig. 1). The duration of tailing
plained below. and the time for complete inactivation of L. monocytogenes
Da-values and Db-values were calculated by taking the neg- were quantified by the parameters ttail and tdl, respectively
ative reciprocal of the slope of the first and the second inactivation (Table 2). Previous studies evaluating the effect of pro-
phase, respectively. In all cases, the duration of first inactivation cessing and storage of fermented foods on the survival of
phase was greater than the Da-value (ta ⬎ Da), thus indicating L. monocytogenes have also shown that inactivation of the
that during ta, log reductions exceeded 1 Da. Therefore, the num-
pathogen is often characterized by extended tailing (6, 33).
ber of decimal reductions (NDa ) during the first inactivation phase
was estimated by the following formula (equation 2):
This phenomenon has been explained by the ability of bac-
terial cells to adapt to environmental stresses, such as acid,
ta heat, anaerobiosis, and nutrient starvation (26), through var-
NDa ⫽ (2)
Da ious cellular mechanisms (24, 45). In a recent study on the
For a specific n-log reduction (nD), e.g., a 2- or 4-log reduction, acid tolerance of L. monocytogenes on frankfurters formu-
the ‘‘remaining D-value’’ (RD) was estimated by equation 3: lated with antimicrobials and exposed periodically to sim-
ulated gastric fluid (pH 1.0), it was concluded that pathogen
RD ⫽ nD ⫺ NDa (3)
resistance was dependent on the initial pathogen numbers
When the RD was ⬍0, then the whole nD reduction had occurred on the product as well as sufficient storage time for survi-
within the ta, and it was characterized by the Da; therefore, it was vors to develop acid resistance (37). Specifically, surviving
calculated by formula 4: population of L. monocytogenes on frankfurters formulated
tnD ⫽ ta ⫹ (RD ⫻ Db) (4) with 0.25% sodium diacetate and dipped in 2.5% lactic acid
(final pH, approximately 5.5) developed marked acid resis-
When RD was ⬎0, a portion of the nD reduction had occurred
during the second phase of the inactivation curve, and it was char- tance within 20 days of storage at 10⬚C (37). Furthermore,
acterized by the Db; therefore, it was calculated by formula 5: it is known that bacterial cells become more resistant to
stress factors when they are attached to the surface of a
tnD ⫽ ta ⫹ (RD ⫻ Db) (5)
product compared with planktonically growing cells (22).
To determine differences between the initial populations (in- The parameters a, ta, b, t2D, t4D, tdl, and ttail were used to
oculum level) and to further enhance the direct comparison of the quantify the effect of examined factors—storage tempera-
inactivation curves, two more parameters were estimated: the time ture, packaging type, and inoculum level—on the biphasic
needed for the L. monocytogenes population to reach the detection inactivation of L. monocytogenes. Specifically, a three-way
limit (tdl) (2 log CFU/g) and the duration of extended survival analysis of variance indicated that all the factors and their
(ttail) (noncountable cells, but the presence of pathogen by enrich-
interactions had significant (P ⬍ 0.05) effects on the in-
ment) before complete elimination of the pathogen (absence of
pathogen by enrichment). activation of L. monocytogenes.
To validate the effect of storage temperature, packaging, and With regard to storage temperature, our results clearly
inoculum level on the survival of L. monocytogenes, individual demonstrated that the reduction of L. monocytogenes was
parameters (a, b, Nb, ta, t2D, t4D, tdl, and ttail) were used as de- greater at room temperature (25⬚C) than at 15⬚C and chill
pendent variables for a mean comparison. The parameters were (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

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

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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 contribute to the improvement of guidelines for the storage
(10) showed enhanced survival of L. monocytogenes and of packaged or unpackaged fermented salami.
E. coli O157:H7 during storage of Lebanon bologna slices The initial inoculation level also appeared to affect (P
(pH 4.6) at 3.6⬚C compared with storage at 13⬚C. Although ⬍ 0.05) the duration and rate of the first inactivation phase
indicative of the potential temperature effects on the fate of of L. monocytogenes (Table 1). More specifically, high in-
L. monocytogenes, results of the above-mentioned studies oculum (6.5 log CFU/g) resulted in a shorter t2D (faster
are not comparable because of the large diversity in process destruction) than low inoculum (4.6 log CFU/g; Table 2).
parameters and product characteristics (e.g., pH, water ac- In addition, although numbers of L. monocytogenes were
tivity, variations in time and temperature). reduced below the detection limit in all cases, the time
The type of packaging in combination with storage needed for a reduction to the detection limit (tdl: F ⫽ 91.03,
temperature affected (P ⬍ 0.05) the duration of the first P ⬍ 0.05) and complete elimination (ttail: F ⫽ 852.24, P
inactivation phase (ta), as well as the destruction rate (b) of ⬍ 0.05) was longer for the high inoculum (Table 2). Similar
the second inactivation phase (Table 1). More specifically, findings have been reported by Yoon et al. (44), who ob-
vacuum packaging resulted in the slower destruction of L. served that high levels (6.8 log CFU/cm2) of E. coli O157:
monocytogenes than aerobic packaging, and this effect in- H7 experienced faster inactivation than low levels (3.8 log
creased as storage temperature decreased (Fig. 1). Our re- CFU/cm2) during drying of beef jerky that had been sub-
sults are consistent with results of previous studies that jected acidic marinade (pH on meat surface, 4.23) before
demonstrated that the storage of salami (15) or pepperoni drying. It has also been reported that stationary-phase
slices (16) at ambient temperature (21⬚C) or aerobic con- gram-negative cells of low density are more sensitive to
ditions was more effective at reducing levels of E. coli low pH than those of high cell density (12). On the other
O157:H7 than refrigeration (4⬚C) or storage under vacuum. hand, Buchanan et al. (4) observed that inactivation rates
Similarly, it has been reported that aerobic conditions re- of L. monocytogenes in liquid media were independent of
sulted in higher destruction of L. monocytogenes in simu- initial population density. However, the differences between
lated salami matrix than under vacuum at 20⬚C (42). How- our findings and those of Buchanan et al. are likely asso-
ever, in the same study, storage at 4⬚C under vacuum re- ciated with differences in the number and type of L. mon-
sulted in slightly higher destruction of L. monocytogenes ocytogenes strains as well as in the type of media (liquid
than in aerobic storage (42). In general, storage recommen- media versus food matrix). More specifically, in the study
dations for salami suggest refrigeration for packaged prod- of Buchanan et al., a mixture of three strains of L. mono-
ucts for an indefinite period and for only 3 weeks, once the cytogenes (Scott A, HO-VJ-S, and V-7) was inoculated in
products are opened (39). The present study indicates that liquid media, whereas we used a single L. monocytogenes
vacuum packaging enhances survival of L. monocytogenes Scott A strain on the surface of salami slices. Thus, the
compared with aerobic storage (unpackaged products), es- results of Buchanan et al. likely represent the survival be-
pecially at low temperatures. Therefore, our results may 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-

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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.

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