ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 25 (2008) 313–323
www.elsevier.com/locate/fm

Microbial ecology of food contact surfaces and products of small-scale

facilities producing traditional sausages
Antonia S. Gounadakia, Panagiotis N. Skandamisa,b,,
Eleftherios H. Drosinosb, George-John E. Nychasa
a
Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and Technology,
Agricultural University of Athens, Iera Odos 75, Athens 11855, Greece
b
Laboratory of Food Hygiene and Quality Control, Department of Food Science and Technology, Agricultural University of Athens,
Iera Odos 75, Athens 11855, Greece
Received 20 May 2007; received in revised form 29 September 2007; accepted 1 October 2007
Available online 6 October 2007

Abstract

The microbial status in 7 small-scale facilities (SSFs) producing traditional fermented and/or dry sausages was investigated. It was
shown that the hygienic status of the processing environment and equipment plays an essential role in the microbial stability and safety of
the final products. The current study revealed that the majority of the sampling sites (control points) tested were highly (44 log CFU/
cm2) contaminated by spoilage flora (i.e. Pseudomonas, Enterobacteriaceae), with knives, tables and mincing machines being the most
heavily contaminated surfaces. Moreover, Listeria monocytogenes, Salmonella spp. and Staphylococus aureus were detected in 11.7%,
26.4%, and 11.7% of the food contact surfaces, respectively. The presence of these pathogens seemed to be associated with high numbers
of one or more specific groups of the ‘house-flora’ on the sampling sites of the facilities; however, high numbers of ‘house-flora’ do not
always suggest the presence of pathogens. With regard to product samples, batter samples were heavily contaminated with the ‘house-
flora’ present on surfaces and equipment of the processing facilities while by the end of processing (final products) LAB constituted the
predominant microbial flora of all products. The low initial levels of S. aureus and Salmonella found in batter samples as well as the
combination of hurdles (mainly awo0.92, average pH ca. o5.0 and competitive effect of natural flora) in the final products were able to
inhibit and/or eliminate these pathogens; however, the detection of L. monocytogenes in 3 out of the 7 final products examined is
indicative of cross-contamination. Our findings further indicate that inadequate hygiene practices within small-scale-processing facilities
may result in loss of microbial control. Therefore, this study addresses the need for strict control measures within SSFs producing
traditional fermented sausages.
r 2007 Elsevier Ltd. All rights reserved.

Keywords: Microbial ecology; Hygiene practices; Safety control; Traditional sausages

1. Introduction improve food safety management systems (e.g. Hazard

Traditional deli meats, such as fermented and/or dry
sausages, are increasingly popular and represent a sig-
nificant proportion of the food market. This worldwide
trend has forced traditional producers to adapt and
Corresponding author. Laboratory of Microbiology and Biotechnol-
ogy of Foods, Department of Food Science and Technology, Agricultural
University of Athens, Iera Odos 75, Athens 11855, Greece.
Tel.: +30 210 5294684; fax: +30 210 5294683.
E-mail address: pskan@aua.gr (P.N. Skandamis).
Analysis and Critical Control Point, HACCP), as well as to
comply with the imposed official food safety regulations in
order to provide safe and quality products to today’s
health-conscious consumers. Indeed, generally accepted
prerequisite programmes such as Good Manufacturing
Practices (GMPs), Good Hygiene Practices (GHPs), and
Standard Operating Procedures (SOPs) could provide the
foundation for effective HACCP implementation, in terms
of the basic environmental and operating conditions that
are necessary for the production of safe food (Maunsell
and Bolton, 2005). However, the management systems

0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fm.2007.10.001
 ARTICLE IN PRESS
314 A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323
mentioned above is not an easy task for small-scale
producers, who experience difficulties in complying with
the prerequisite programmes generally adopted from large
processing plants (Mossel et al., 1998). This lack of control
programs within traditional facilities producing fermented
and/or dry sausages is of particular concern for product’s
safety, especially when their production relies on natural
microbial populations originating from raw materials and
processing ‘house-flora’ or otherwise stated ‘resident
microflora’ (Leriche et al., 2003; Chevallier et al., 2006).
Moreover, evidence exists that inadequate hygiene
practices within food processing plants may result in the
contamination of product with pathogens (Metaxopoulos
et al., 2003) and therefore pose a subsequent risk in the
product’s safety. On the other hand, complete elimination of
pathogens from raw materials (Eisel et al., 1997) and food
processing environment (Tompkin, 2002) is difficult, parti-
cularly when many food pathogenic are known to be able to
attach on food contact surfaces (Fonnesbech-Vogel et al.,
2001; Jessen and Lammert, 2003; Deza et al., 2005) and
remain viable even after cleaning and disinfection (Frank
and Koffi, 1990). Therefore, evaluation of the presence and/
or the level as well as the control of technological, spoilage
and pathogenic microorganisms is essential in order to
produce fermented and/or dry sausages that satisfy the
criteria of hygienic quality, organoleptic characteristics and
food safety (Chevallier et al., 2006).
Available studies exist for the evaluation of the effec-
tiveness of currently applied hygienic practices within high-
throughput food producers such as deli-meat producers.
Numerous such studies have demonstrated that product
contamination may originate from the processing environ-
ment (Eisel et al., 1997; Samelis and Metaxopoulos, 1999).
However, the majority of these studies have focused either
on monitoring the prevalence of specific pathogens such as
Listeria monocytogenes, Escherichia coli O157:H7 and
Salmonella spp. (Lunden et al. 2003a, b; Peccio et al.,
2003; Holan et al., 2004) or on specific spoilage bacteria
such as Pseudomonas spp. within the processing environ-
ment and equipment while the overall microbial ecology of
the food processing environment has not been fully
elucidated. Several other studies have dealt with the
microbial ecology of various meat products rather than
on equipment, as an attempt to indicate possible micro-
biological risks (Samelis and Metaxopoulos, 1999; Drosi-
nos et al., 2005; Murphy et al., 2005; Trevenot et al., 2005).
Taking into account the fact that the prevalence of
pathogens is strongly associated with the ‘house-flora’ of
the premises (Tompkin, 2002; Carpentier and Chassaing,
2004), there is a need for further analytical information on
possible contamination sources within the processing
environment as part of the microbiological hazard
identification. Such data will also provide small-scale
traditional deli-meat producers with information feasible
to their processes, regarding the impact and the ability of
current GMP/GHP practices applied in enhancing food
safety control.
Considering the above, the objective of this study was to
survey the global microbial ecology (processing environ-
ment and products) of 7 small-scale facilities (SSFs)
producing traditional fermented and/or dry sausages.
2. Materials and methods
2.1. Collection of samples
Samples were collected from 7 SSFs, producing tradi-
tional fermented and/or dry sausages. Sausages were
mainly manufactured with pork or beef lean, fat, NaCl
and spices, with or without addition of starter cultures. All
facilities were divided into 3 main areas: reception of raw
materials, product processing, and product’s storage and
handling. Surface samples were collected from 6 control
points (CPs) of the processing environment and on
equipment from each small-scale facility: 3 from machines
(mincing, mixing, and stuffing), 1 from the cutting table, 1
from wall of the storage cold room, and 1 from knives.
Food-contact surface sampling was performed by swab-
bing a delimited area (5  100 cm2) according to the ISO
18593 (2004) suggested protocol, after the scheduled
cleaning and/or disinfection procedures. More specifically,
swabbing was carried out with a sterile wet cloth
(40 cm  40 cm; Laboratories Humeau, France), containing
15 ml of a neutralizing solution (10% v/v). From each CP 5
swabs were taken, 3 (3  100 cm2; n ¼ 3) for the enumera-
tion of technological and spoilage flora and 2 (2  100 cm2)
for detection of L. monocytogenes (n ¼ 1) and Salmonella
spp. (n ¼ 1). Immediately after swabbing, all samples were
stored at 4 1C and analyzed within 24 h.
Triplicate product samples were taken at different phases
of the processing: (a) batter samples: just after stuffing, (b)
1-week sausages: after fermentation period, and (c) final
products: at the time of commercialization. More specifi-
cally, at each sampling phase, 3 sausages (100 g) from the
same batch were taken and stored at 4 1C and analyzed
within 24 h. From each sample (100 g), sub-samples of
25 g were used for the microbiological analysis of
technological and spoilage flora as well as the enrichment
of L. monocytogenes and Salmonella spp. as described in
the next paragraph.
2.2. Microbiological analysis
For environmental samples, wet cloths were transferred
to stomacher bags containing 25 ml sterile buffered
peptone water solution (BPW, Merck) and macerated for
1 min in a stomacher (Lab Blender 400, Seward Medical,
London). For meat products, microbiological testing was
conducted by the aseptic transfer of 25 g portions to
separate stomacher bags containing 225 ml sterile 0.1%
peptone water with salt (NaCl, 0.85%, w/v). Samples were
then macerated for 1 min in a stomacher (Lab Blender 400,
Seward Medical, London). For environmental and sausage
samples, serial dilutions were made in 9 ml quarter strength
 ARTICLE IN PRESS
A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323
Ringer’s solution (Merck, Germany). In order to enumer-
ate the flora, 1 or 0.1 ml portions of appropriate dilution
were poured or spread onto duplicate plates of the
appropriate culture medium.
Total viable count (TVC) were obtained from the
following plate count agar (PCA, Merck), incubated at
30 1C for 48–72 h; lactic acid bacteria (LAB) on the de
Man, Rogosa, Sharpe agar (MRS, Merck), incubated at
30 1C for 48–72 h; Pseudomonas spp. on Cetrimide–
Fucidin–Cephaloridine (CFC; Merck) agar, incubated at
25 1C for 48 h; Coagulase negative Staphylococcus and
Kocuria on Mannitol Salt red phenol agar (MSA, Merck),
incubated at 30 1C for 72 h; Enterobacteriaceae on crystal
Violet neutral Red Bile Glucose Agar (VRBG, Merck),
incubated at 37 1C for 24 h; Enterococci on Enterococcus
Selective Agar acc. To Slanetz and Batley Agar (ME,
Merck); yeasts and moulds on Rose Bengal Chloramphe-
nicol agar (RBC, Oxoid), incubated at 25 1C for 3–5 days;
Staphylococcus aureus on Baird–Parker agar supplemented
with Tellurite Yolk Egg (BP, Merck), incubated at 37 1C
for 48 h. Colonies showing lecithinase activity on Baird–
Parker medium were tested for coagulase, using rabbit
plasma with EDTA (Biomerieux, France).
2.3. Enrichment procedures for L. monocytogenes and
Salmonella spp.
For L. monocytogenes, a primary enrichment was
performed by suspending 25 g of product samples or the
whole wet cloth in 225 or 25 ml of 1/2 FRASER Listeria
selective enrichment broth (Merck), respectively. Following
incubation at 30 1C for 24 h (primary enrichment), 0.1 ml
portion of samples were transferred to 10-ml FRASER
Listeria selective enrichment broth (Merck) and incubated
at 35 1C for 48 h (secondary enrichment). After each
enrichment step the culture was streaked onto both
PALCAM Listeria agar plates (30 1C for 48 h) (Merck)
and Listeria Agar acc. to Ottaviani and Agosti plates
(37 1C for 24–48 h) (ALOA, Biolife). Presumptive Listeria
colonies isolated either from ALOA (blue-turquoise
colonies with or without a precipitation halo) agar plates
or from PALCAM Listeria agar plates were confirmed
using biochemical tests. Specifically, suspect colonies were
streaked onto Columbia blood agar (Oxoid, UK) and
incubated at 37 1C for 24 h. b-haemolytic colonies were
further tested for motility at 25 1C, catalase reaction, and
carbohydrate acid production from rhamnose (0.5%,
Sigma), xylose (0.5%, Sigma), mannitol (0.5%, Sigma),
and a-methyl-D-mannoside (0.5%, Sigma) in purple carbo-
hydrate broth base (Difco, Becton Dickinson).
For Salmonella spp., enrichment was performed by
suspending 25 g of product samples or the whole wet cloth
in 225 or 25 ml of BPW solution, respectively. Initial
suspensions of product and environmental samples were
incubated at 37 1C for 24 h. Following incubation, 3 drops
were spotted onto the surface of modified Rappaport–
Vasiliadis medium (MRSV, supplemented with MRSV
315
selective supplemented, Merck). After incubation at 42 1C
for 24 h, the plates were observed for motility (opaque
halo). Presumptive Salmonella colonies were streaked onto
Brilliant-green Phenol-red Lactose Sucrose Agar plates
(BPLS, Merck) and incubated at 37 1C for 24–48 h.
Suspected Salmonella colonies were isolated and following
preliminary testing (Gram-staining, oxidase) were further
confirmed by a rapid latex agglutination test (Microgen,
Bioproducts, Surrey, UK).
2.4. Physicochemical analysis
The pH was measured by immersing the pH probe of a
digital pH meter (pH 691, Metrohm) in a diluted and
homogenized sample containing 10 g of sausage and 90 ml
of distilled water. The aw was measured with a calibrated
electric hygrometer (HygroLab, Rotronic, Bassersdorf,
Switzerland) using a sausage sample of 5 g, according to
the manufacturer’s instructions. pH and aw measurements
were performed in triplicates.
2.5. Statistical analysis
The microbiological testing (enumeration of technologi-
cal and spoilage flora) of all samples (CPs of the processing
environment and equipment and products samples) was
performed using triplicate samples. Bacterial counts were
converted in log CFU/g for sausage samples and in
log CFU/cm2 for environmental samples and further
subjected to analysis of variance using the GLM procedure
of XLSTAT statistical program (version 2006.06; Addin-
soft, France). Tukey’s multiple-range test was used to
discriminate means. Means were considered to be signifi-
cantly different at the 95% significance level.
Triplicate samples were also used for the determination
of pathogens (presence/absence) in 25 g of samples
throughout processing. The results were reported as the
number of positive (presence; 3/3) or negative (absence;
0/3) out of the total number of samples from the 7 facilities.
3. Results
3.1. Characterization of the SSFs
A total of 55 samples (34 from CPs of the processing
surfaces and equipment and 21 product samples) were
collected from 7 (SSF 1–7) producing fermented and/or dry
sausages. The majority (6/7) of these SSFs produced
naturally fermented and ripened sausages (Table 1);
however, variation in the process conditions (duration
and temperature of fermentation and/or ripening condi-
tions) between facilities was observed (Table 1). Sampling
of the processing surfaces and equipment was performed
after the cleaning and/or disinfection procedures. The
majority (5/7 or 71.4%) of the examined SSFs used
domestic disinfectants (such as chlorine) for cleaning
processing surfaces and equipment, whereas only 42.8%
 ARTICLE IN PRESS
316 A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323

Table 1
Characteristics of the small-scale facilities (SSFs) producing traditional fermented sausages

Small-scale facility 1 2 3 4 5 6 7

Small-scale facility characteristics

Facility on the farm N N N Y Y N Y
Collective facility Y N N N N N N
Butcher on site N Y Y N N Y N
Temperature (1C) within the facility 12 10 17 12 13 15 15
HACCP Y N Y N Y N N
Approved facility by competent authorities Y N Y Y Y N Y
The establishment is producing in compliance of some GHPsa Y N Y N Y N Y
Use of disinfectant/sanitizer Y N N N Y N N
Process characteristics
Mix processing
Mincing machine Y Y Y Y Y Y Y
Cutter Y N N N N N N
Addition of starter culture Y N N N N N N
Addition of sugar Y N N N N N N
Addition of nitrite salt Y N N N N N N
Inoculation of the surface with moulds N N N N N N N
Inhibitors of mould growth N N N N N N N
Fermentation period
Fermentation temperature 1C 20 9 17 12 15–25 15 15
Length of fermentation (days) 7 5 5 3 3 3 2
Drying/maturation period
Natural dryer N Y Y Y Y N Y
Conditioned dryer Y N N N N Y N
Drying temperature 1C 14 12 17 12 15–25 15–25 15–25
Smoking Y N N N N Y Y
Length of drying (weeks) 7 3 2 2 2–3 2–3 2–3
Packaging V/A A A A A A A

Y: yes; N: no; V: vacuum; A: air.

a
Good Hygiene Practices.

(3/7) of the plants had a hazard analysis system in place
(Table 1).
3.2. Microbiological analysis of the processing environment
Considering the house-flora, the microbial counts varied
(Po0.05) with the sampling site (CP) and facility (Table 2).
Specifically, LAB were detected in all of the CPs sampled,
with their counts ranging between o0.30 (detection limit)
and 7.43 log CFU/cm2, depending on the sampling site
(Po0.05; Table 2). High LAB counts (45 log CFU/cm2)
were recovered from 42.8% (3/7) of knives, 57.1% (4/7) of
cold rooms’ surfaces and from 33.3% (1/3) of mixing
machines whereas counts in all (3) stuffing machines and in
57.1% (4/7) of mincing machines and tables ranged
between 2 and 5 log CFU/cm2 (Table 2). In any case,
LAB counts within each CP sampled differed significantly
(Po0.05) between the majority of facilities (Table 2). The
highest LAB counts (7.43 log CFU/cm2) were recovered
from the mixing machine of SSF 7 (facility operating
without HACCP), whereas the lowest counts were recov-
ered from all the sampling sites of SSF 1 and the cold room
of SSF 3 (both facilities having HACCP; Tables 1 and 2).
While Staphylococci/Kocuria were detectable in 82.3%
(28 of 34) of the environmental samples, significant
variation (Po0.05) in their counts was observed in
specific sites of different facilities (Table 2). Their counts
reached 43 log CFU/cm2 in the 85.7% (6/7) of mincing
machines as well as in 71.43% (5/7) of mixing machines,
knives and tables and the 33.3% (1/3) of stuffing
and mixing machines, respectively (Po0.05; Table 2).
The highest counts of Staphylococci/Kocuria (7.6 log
CFU/cm2) were recovered from the mincing machine
of SSF 6 (facility operating without HACCP; Tables 1
and 2).
Counts of enterococci were below the detection limit
(o0.30 log CFU/cm2) in 17.6% (6/34) of samples from the
mincing machine, the knife of SSF 1 as well as the tables of
SSF 1 and 4 and the cold rooms SSF 3 and 4 (Table 2). In
the majority (57.8%) of environmental samples, the levels
of enterococci were uniformly distributed between 1 and
4 log CFU/cm2 (Table 2). However, high levels of enter-
ococci (44 log CFU/cm2) were recovered from 3 mincing
machines (SSF 2, 3, and 5), 2 knives (SSF 3 and 4; Po0.05)
as well as from the tables of SSF 2 and 6 (Po0.05) and the
cold room of SSF 7 (Table 2).
 ARTICLE IN PRESS
A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323 317

Table 2
Counts (mean log CFU/cm2 and standard deviation (SD); n ¼ 3) of the observed microbial association on specific sampling sites within the processing
environment of different small-scale facilities (SSFs)

Control Facility LAB Staphylococcus/ Yeast and moulds Enterococcus Enterobacteriaceae Pseudomonas TVCa
point identification Kocuria

Mincing 1 o0.30 Ec (0.00)* o1.30 Eb (0.00)* 2.45 Ea (0.21) o0.30 Ec (0.00)* o0.30 Fc (0.00)* o1.30 Eb (0.00)* 3.00 Fa (0.00)
machines 2 4.90 Cc ( 0.11) 3.54 Dd (0.02) 2.84 Ee (0.09) 4.84 Bc (0.03) 6.58 Aa (0.04) 3.60 Cd (0.09) 6.18 Eb (0.02)
3 3.51 De (0.03) 4.99 Cb (0.12) 3.32 Df (0.03) 4.69 Bc (0.12) 3.10 Eg (0.01) 3.73 Cd (0.05) 7.25 Da (0.03)
4 3.70 Dd (0.04) 5.75 BCb (0.21) 4.56 Cc (0.04) 1.96 Ce (0.26) 5.38 Cb (0.04) 7.20 Aa (0.07) 7.27 Da (0.02)
5 3.55 Dd (0.01) 5.19 Cc (0.16) 3.32 Dde (0.09) 5.41 Abc (0.10) 5.61 Bb (0.02) 3.13 De (0.18) 7.59 Ca (0.02)
6 6.48 Ac (0.02) 7.60 Ab (0.43) 5.13 Bd (0.00) 1.30 De (0.00) 4.98 Dd (0.02) 7.44 Ab (0.01) 8.88 Ba (0.03)
7 5.53 Bc (0.07) 6.44 Bb (0.09) 6.19 Ab (0.11) 2.30 Ce (0.00) 5.37 Ccd (0.10) 5.11 Bd (0.02) 9.25 Aa (0.01)

Mixing 4 2.93 Bf (0.06) 5.60 Bc (0.00 ) 3.94 Bd (0.06) 3.00 Bf (0.00) 3.39 Ce (0.03) 5.87 Ab (0.02) 7.48 Ca (0.02)
machines 5 o0.30 Cf (0.00)* 5.24 Bd (0.14) 5.39 Acd (0.15) 3.59 Ae (0.08) 6.50 Ab (0.03) 5.76 Ac (0.14) 7.58 Ba (0.03)
7 7.43 Ab (0.11) 6.29 Ac (0.19) 5.47 Ad (0.01) 1.30 Cf (0.00) 3.88 Be (0.11) 3.56 Be (0.03) 8.28 Aa (0.02)

Stuffing 1 2.36 Bb (0.08) o1.30 Cd (0.00)* o1.30 Cd (0.00)* 2.14 Bbc (0.09) 1.84 Bc (0.34) 2.54 Bab (0.34) 2.90 Ca (0.14)
machines 3 2.56 Bbc (0.03) 2.75 Bab (0.21) 2.69 Bb (0.12) 1.84 Bde (0.09) 2.30 Bd (0.00) o1.30 Ce (0.00)* 4.48 Ba (0.07)
5 4.46 Ae (0.08) 5.69 Ac (0.12) 5.46 Ac (0.08) 2.60 Af (0.00) 4.87 Ad (0.08) 8.41 Ab (0.03) 8.70 Aa (0.02)

Knives 1 o0.30 Ge (0.00)* o1.30 Dd (0.00)* 4.34 Ca (0.03) o0.30 Ee (0.00)* 3.85 Dc (0.05) 4.06 Ebc (0.01) 4.18 Eab (0.26)
2 4.89 Dc (0.08) 6.02 Ab (0.34) o1.30 Ef (0.00)* 3.85 Cd (0.10) 5.64 Cb (0.04) 2.80 Fe (0.28) 7.46 Ca (0.03)
3 2.30 Fd (0.00) 2.45 Cd (0.21) 3.05 Dc (0.21) 4.30 Bb (0.00) o0.30 Ef (0.00)* o1.30 Ge (0.00)* 6.75 Da (0.08)
4 6.40 Ac (0.03) 4.60 Be (0.00) 6.94 Ab (0.02) 4.89 Ad (0.16) 6.80 Bb (0.01) 9.00 Aa (0.02) 9.04 Aa (0.05)
5 5.55 Bd (0.01) 5.63 Acd (0.07) 5.80 Bc (0.07) 3.60 Ce (0.00) 5.64 Ccd (0.02) 6.70 Cb (0.08) 8.23 Ba (0.03)
6 3.97 Ed (0.04) 4.15 Bd (0.00) 4.59 Cc (0.39) 1.30 De (0.00) 3.97 Dd (0.04) 5.90 Db (0.03) 6.42 Da (0.02)
7 5.37 Cb (0.01) 4.75 Bbc (0.21) 4.14 Cc (0.08) 1.30 Dd (0.00) 8.28 Aa (0.02) 8.25 Ba (0.00) 8.81 Aa (0.01)

Tables 1 o0.30 Fd (0.00)* o1.30 Dc (0.00)* 3.02 Da (0.15) o0.30 Ed (0.00)* 1.77 Fb (0.33) 3.15 Ca (0.63) 3.45 Da (0.53)
2 5.14 Bd (0.06) o1.30 Df (0.00)* 4.53 BCe (0.03) 4.58 Ae (0.03) 5.53 BCc (0.01) 6.45 Bb (0.01) 7.43 BCa (0.03)
3 3.06 Ed (0.11) 5.75 Aab (0.21) 3.08 Dd (0.25) 1.30 De (0.00) 3.71 Ebc (0.02) 3.36 Ccd (0.08) 7.14 Ca (0.03)
4 4.47 Cf (0.00) 6.04 Ac (0.06) 5.16 Ae (0.05) o0.30 Eg (0.00)* 5.79 ABd (0.02) 8.29 Ab (0.00) 8.85 Aa (0.06)
5 4.21 De (0.06) 5.19 Bd (0.16) 4.17 Ce (0.05) 3.23 Cf (0.04) 6.49 Ac (0.05) 6.78 Bb (0.00) 8.35 ABa (0.03)
6 5.89 Ac (0.02) 4.30 Ce (0.00) 4.94 ABd (0.08) 4.32 Be (0.03) 4.79 Dd (0.10) 8.26 Ab (0.01) 9.15 Aa (0.02)
7 2.99 Ee (0.04) 4.99 Bc (0.12) 3.37 Dd (0.16) 1.30 Df (0.00) 4.96 Dc (0.05) 5.98 Bb (0.00) 8.36 ABa (0.06)

Cold 1 o0.30 Ff (0.00)* 3.75 Ca (0.04) 2.89 Eab (0.16) 2.23 Cde (0.04) 2.54 Fcd (0.05) 2.69 Dbc (0.12) 2.80 Ebc (0.28)
rooms 2 5.26 Da (0.07) 3.04 Dc (0.06) 5.15 Cab (0.02) 2.37 Cd (0.10) 2.96 Ec (0.02) 5.32 Ba (0.03) 4.98 Db (0.02)
3 o0.30 Fc (0.00)* 6.17 ABa (0.12) o1.30 Fb (0.00)* o0.30 Ec (0.00)* o0.30 Gc (0.00)* o1.30 Eb (0.00)* 7.18 BCa (0.02)
4 3.15 Ed (0.05) o1.30 Ee (0.00)* o1.30 Fe (0.00)* o0.30 Ef (0.00)* 4.07 Cc (0.04) 4.68 Cb (0.01) 6.87 Ca (0.02)
5 5.55 Cc (0.07) 6.25 ABb (0.24) 4.55 Dd (0.03) 2.63 Bf (0.04) 3.76 De (0.04) 5.74 Ac (0.01) 7.38 Ba (0.01)
6 6.28 Bc (0.04) 6.29 Ac (0.00) 7.35 Bb (0.02) 1.30 De (0.00) 5.66 Bd (0.05) 5.80 Ad (0.08) 7.92 Aa (0.02)
7 6.60 Ab (0.03) 5.83 Bd (0.04) 7.83 Aa (0.12) 4.68 Af (0.05) 6.28 Ac (0.02) 5.23 Be (0.05) 7.92 Aa (0.01)

*Counts below the detection limit of the method; however for the means of statistical analysis the value of the detection limit (either 0.30 or 1.30) was used
for each microbial group. Within each row, underlined values are indicative of the dominant microorganism confirmed at significance level of 0.05.
abcdefg: means within a row sharing at least a common letter are not significantly different (Po0.05).
ABCDEFG: for each CCP, means within a column sharing at least a common letter are not significantly different (Po0.05).
a
Total viable count.

Counts of Enterobacteriaceae ranged between 4 and
9 log CFU/cm2, in 71.4% (5/7) of mincing machines and
tables as well as the stuffing and mixing machines of SSF 5
(Po0.05; Table 2). Such high levels were also recovered
from 57.1% (4/7) to 42.8% (3/7) of knives and cold rooms,
respectively (Po0.05; Table 2). The levels of Pseudomonas
as well as of yeasts and moulds also ranged between 4 and
9 log CFU/cm2, in 21 and 19 of all (34) environmental
samples tested, respectively (Po0.05; Table 2). Specifically,
the levels of Pseudomonas exceeded 7 log CFU/cm2 in the
stuffing machine of SSF 5, the mincing machines and the
tables of SSF 4 and 6 (P40.05) as well as the knives of SSF
4 and 7 (Po0.05; Table 2), whereas similar levels of yeasts
and moulds were recovered from the cold rooms of SSF 6
and 7 (Po0.05; Table 2).
Considering the hygienic status of the SSFs, TVC were
used to compare the overall contamination levels among
the seven facilities. In general, the majority (27/34) of the
environmental samples were characterized by high con-
tamination levels (TVC levels of 46 log CFU/cm2) while
the statistical analysis revealed that in the 51.8% (14/27) of
these samples Pseudomonas were the predominant micro-
organisms followed by Enterobacteriaceae, yeasts and
moulds and Staphylococci/Kocuria (Table 2). With regard
to specific sampling sites, the knives of SSF 4–7, the tables
and of SSF 2, 4–6 as well as the mincing machines of SSF 4
and 6 were among the most heavily contaminated surfaces
(Table 2). Regardless of sampling site, SSF 1 was always
characterized by the lowest TVC levels (facility operating
with HACCP), while the highest TVC levels were recovered
 ARTICLE IN PRESS
318 A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323

Table 3
Overall prevalence and distribution of pathogenic bacteria on specific sampling sites within the processing environment

Control points Samples containing unsatisfactory levels (%)

L. monocytogenes Salmonella spp. S. aureus

a b c a
Positive samples SSFID % Samples Positive samples SSFIDb % Samples c
Positive samplesa SSFIDb % Samplesc

Mincing machines 1/7 (14.29) 4 2.94 2/7 (28.57) 2; 5 5.88 1/7 (14.29) 4 2.94
Mixing machines 0/3 (0.00) – 0.00 0/3 (0.00) – 0.00 1/3 (33.33) 4 2.94
Stuffing machines 1/3 (33.33) 5 2.94 0/3 (0.00) – 0.00 1/3 (33.33) 5 2.94
Knives 1/7 (14.29) 2 2.94 3/7 (42.86) 2; 5; 7 8.82 0/7 (0.00) – 0.00
Tables 0/7 (0.00) – 0.00 1/7 (14.29) 3 2.94 1/7 (14.29) 2 2.94
Cold rooms 1/7 (14.29) 6 2.94 3/7 (42.86) 2; 5; 6 8.82 0/7 (0.00) – 0.00
Total 4/34 11.76 9/34 26.46 4/34 11.76
a
% Distribution per control point.
b
Small-scale facility identification.
c
Overall samples.

Table 4
Changes (mean and standard deviation (SD); n ¼ 3) in product characteristics (pH, aw) at different manufacturing stages of the examined products

Small-scale facility 1 2 3 4 5 6 7

pH
Batter 5.68 Ab (0.06) 6.08 Ba (0.00) 4.48 Ce (0.01) 4.91 Ad (0.02) 4.92 Ad (0.02) 5.34 Bc (0.01) 5.28 Ac (0.01)
1-week sausage 4.66 Bc (0.16) 6.11 Aa (0.00) 4.76 Bc (0.06) 4.78 Ac (0.11) 4.71 Bc (0.01) 5.25 Cb (0.01) 5.26 Ab (0.02)
Final product 4.06 Cd (0.06) 6.04 Ca (0.00) 4.92 Ac (0.04) 4.73 Ac (0.01) 4.73 Bc (0.01) 5.72 Ab (0.01) 4.72 Bc (0.12)
aw
Batter nd nd nd nd nd nd nd
1-week sausage nd nd nd nd nd nd nd
Final product 0.865 d (0.01) 0.925 a (0.02) 0.915 ab (0.01) 0.885 cd (0.02) 0.905 abc (0.01) 0.920 ab (0.02) 0.895 bc (0.02)

nd: not determined.

abcdefg: means within a row sharing at least a common letter are not significantly different (Po0.05).
ABCDEFG: for pH readings, means within a column sharing at least a common letter are not significantly different (Po0.05).

from the processing environment of SSF 5 (facility
operating with HACCP) and SSF 4, 6, and 7 (facilities
operating without HACCP) (Po0.05; Table 2).
With regards to pathogenic flora, Salmonella was
isolated from 4 sampling sites at a rate of 26.46% of the
samples, mainly from 5 facilities (Table 3). Similarly,
L. monocytogenes was present at 4 sampling sites of 4
facilities resulting in an overall frequency of 11.7% over the
total samples (Table 3). S. aureus was also found to persist
in the environment of 3 facilities and it was isolated
from 4 sampling sites at the same frequency as that of
L. monocytogenes (Table 3).
3.3. Microbiological analysis and physicochemical
characteristics of the sausages
With regards to the physicochemical characteristics of
products, significant variation (Po0.05) in the pH of
the batter samples was observed between different SSFs
(Table 4). Specifically, in the majority (4/7) of final
products, the pH varied from 4.72 to 4.92 (P40.05),
whereas in 2 of the final products the pH was above of 5.3
(Table 4). In addition, significant variation (Po0.05) in the
levels of water activity (aw) was observed among final
products ranging between 0.92 and 0.87 (Table 4).
Regarding the microbial flora of the products, LAB
levels ranged between 4 and 8 log CFU/g in all batter
samples (Table 5). By the end of fermentation (1-week
sausage), LAB levels exceeded 8 log CFU/g (P40.05) in
71.4% (5/7) of products (Table 5; Fig. 1). Conversely their
counts remained stable or slightly increased during
ripening (end product) (Table 5) constituting the predomi-
nant microbial flora of all products (Table 5; Fig. 1).
Significant variation (Po0.05) in the counts of Staphy-
lococci/Kocuria was observed (Table 5). Their population
ranged between 3 and 5 log CFU/g in 6 out of 7 batter
samples; however, their levels declined below 2 log CFU/g
in 2 and in 5 of the products by the end of fermentation
and ripening, respectively (Table 5; Fig. 1).
Enterococci were uniformly distributed between 2 and
6.5 log CFU/g in 4 of the batter samples (Po0.05; Fig. 1),
whereas their counts declined below the detection limit
(o1 log CFU/g) by the end of ripening in 5 out of the 7
products. Pseudomonas and Enterobacteriaceae were re-
covered from all (21) samples and their levels varied
significantly between 2 and 8 log CFU/g(Po0.05; Table 5;
 ARTICLE IN PRESS
A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323 319

Table 5
Changes in the counts (mean log CFU/g and standard deviation (SD); n ¼ 3) of the observed microbial association at different stages during the
production of the examined products

Productsa Facility LAB Staphylococcus/ Yeast and Enterococcus Enterobacteriaceae Pseudomonas TVCb
identification Kocuria moulds

Batter 1 5.82 Bf (0.01) 3.00 Ed (0.00) 3.49 De (0.02) 1.81 Fd (0.05) 3.77 Cd (0.01) 3.79 Cf (0.01) 7.50 Ac (0.14)
2 7.69 Ab (0.00) 4.30 BCbc (0.03) 3.78 Cd (0.00) 2.18 Dd (0.20) 2.35 De (0.49) 4.87 Be (0.12) 8.18 Aa (0.04)
3 7.42 Ac (0.00) 4.12 Fc (0.16) 4.71 Ec (0.05) 6.35 Ca (0.04) 2.93 Ge (0.04) 5.22 Dd (0.11) 6.65 Be (0.00)
4 6.38 Be (0.05) 4.12 Fc (0.00) 4.72 Dc (0.02) o1.00 Ge (0.00)* 4.30 Ecd (0.03) 6.13 Cc (0.00) 7.00 Ad (0.00)
5 8.01 Ba (0.03) 7.13 Ea (0.07) 7.42 CDa (0.01) 5.65 Fb (0.07) 7.38 DEa (0.18) 8.34 Aa (0.04) 7.69 Cbc (0.00)
6 6.98 Ad (0.02) 4.60 Bbc (0.43) 4.95 Bb (0.07) o1.00 Ce (0.00)* 4.83 Bc (0.02) 7.00 Ab (0.03) 6.73 Ae (0.00)
7 4.02 Eg (0.01) 4.94 Db (0.00) 4.79 Dc (0.04) 3.54 Ec (0.34) 5.68 Cb (0.16) 7.00 Bb (0.04) 7.72 Ab (0.00)

1-week 1 8.82 Aa (0.00) 4.39 Dd (0.12) 2.87 Ee (0.12) 2.77 Ec (0.10) 4.91 Ce (0.02) 5.52 Be (0.06) 8.90 Ab (0.09)
sausage 2 8.67 Aa (0.01) o2.00 Ff (0.00)* 2.95 De (0.00) o1.00 Gd (0.00)* 2.30 Ef (0.00) 4.13 Cf (0.07) 7.11 Be (0.05)
3 8.84 Aa (0.11) 4.00 Ee (0.00) 5.30 Db (0.06) 6.48 Ca (0.04) 7.32 Ba (0.16) 7.11 Bb (0.03) 8.87 Ab (0.06)
4 7.80 Bb (0.01) o2.00 Ff (0.00)* 5.04 Ec (0.00) o1.00 Gd (0.00)* 6.79 Cb (0.02) 6.25 Dd (0.00) 8.56 Ac (0.00)
5 8.81 Ba (0.01) 7.90 Ca (0.01) 7.96 Ca (0.07) 6.00 Ea (0.06) 6.53 Dc (0.04) 7.94 Ca (0.06) 9.21 Aa (0.08)
6 7.76 Bb (0.09) 7.17 Cb (0.08) 5.02 Fc (0.02) o1.00 Gd (0.00)* 5.68 Ed (0.04) 6.94 Dbc (0.06) 8.80 Ad (0.12)
7 8.81 Aa (0.09) 6.72 Bc (0.01) 3.98 Ed (0.03) 5.24 Db (0.34) 5.84 Cd (0.02) 6.80 Bc (0.05) 8.04 Abc (0.00)

Final 1 9.18 Ab (0.00) o2.00 Db (0.00)* 5.62 Ec (0.01) 6.84 Ea (0.09) 4.91 Ce (0.02) 8.17 Ba (0.01) 8.73 Ab (0.03)
sausage 2 6.48 Bg (0.01) o2.00 Fb (0.00)* 3.60 Cf (0.00) o1.00 Gc (0.00)* 2.19 Ef (0.02) 3.00 Dd (0.00) 7.90 Ae (0.05)
3 8.73 Ad (0.12) 6.93 Da (0.04) 7.71 Bb (0.04) o1.00 Ec (0.00)* 7.32 Cb (0.16) 7.93 Ba (0.02) 8.25 Ad (0.02)
4 7.80 Bf (0.01) o2.00 Fb (0.00)* 5.11 Ed (0.05) o1.00 Gc (0.00)* 6.83 Cc (0.01) 6.30 Dc (0.00) 8.45 Ac (0.02)
5 8.52 Be (0.02) o2.00 Eb (0.00)* 7.89 Ca (0.08) 6.45 Db (0.21) 6.30 Dd (0.00) 8.09 BCa (0.20) 9.09 Aa (0.04)
6 8.98 Ac (0.02) 6.60 Ca (0.43) 5.08 Dd (0.02) o1.00 Ec (0.00)* 7.83 Ba (0.02) 7.11 Cb (0.04) 8.65 Ab (0.01)
7 10.03 Aa (0.03) o2.00 Eb (0.00)* 4.44 De (0.01) o1.00 Fc (0.00)* 2.00 Ef (0.00) 6.54 Cc (0.04) 8.45 Bc (0.02)

*Counts were below the detection limit of the method; however for the means of statistical analysis the value of the detection limit (either 1 or 2) was used
for each microbial group. Within each row, underlined values are indicative of the dominant microorganism confirmed at significance level of 0.05.
ABCDEFG: Means within a row sharing at least a common letter are not significantly different (Po0.05).
abcdefg: For each product period, means within a column sharing at least a common letter are not significantly different (Po0.05).
a
Batter sample: just after stuffing; 1-week sausage: after fermentation period; and final sausage: product at the time of commercialization.
b
Total viable count.

Fig. 1). More specifically, high levels (X7 log CFU/g) of
Pseudomonas were recovered from the batter samples of
SSF 5–7 while by the end of ripening such high levels were
recovered from the products of SSF 1, 3, 5, and 5 (Table 5).
Moreover, high levels (X4 log CFU/g) of Enterobacteria-
ceae were recovered from the final products of SSF 1, and
3–6. Furthermore, yeasts and moulds were detected in all
samples (Table 5) and their counts varied significantly
between approx. 3 and 8 log CFU/g (Po0.05; Table 5;
Fig. 1).
Sausage samples were also examined for the presence
and distribution of pathogenic bacteria throughout proces-
sing (Table 6). S. aureus was not detected at any sample
(Table 6). Salmonella and L. monocytogenes were recovered
from batter samples of SSF 4 and 5 at a frequency of
14.2% (Table 6). However, Salmonella was not detected at
any final product, whereas L. monocytogenes was detected
in 3 of the final products (Table 6).
4. Discussion
It is widely recognized that GHPs form the basis or a
vital part of HACCP (FAO/WHO, 2004). Under the
current food hygiene regulations, all food businesses within
EU except for primary producers are required to operate
food safety management procedures based on HACCP
principles (EU Directive EC No. 852/2004). However,
small-scale businesses may lack the in-house knowledge
and resources to correctly implement HACCP. Indeed, the
majority of the SSFs studied (Table 1) operated without an
HACCP system in place, using insufficient sanitation
procedures (i.e. using solely rinsing with water or domestic
cleaners). An insufficient cleaning and disinfection may not
remove all meat or fat residues, and these residues may act
as vehicles for spoilage or pathogenic bacteria from
surfaces to meat (Gill and McGinnis, 2004) and thereafter
to sausages.
It has been demonstrated that the ‘house-flora’ of
facilities producing traditional fermented and/or dry
sausages includes useful microorganisms for the fermenta-
tion (e.g. LAB) and the flavour of sausage (i.e. Staphylo-
cocci/Kocuria), as well as spoilage (i.e. Pseudomonas) and
pathogenic flora (i.e.. L. monocytogenes, S. aureus)
(Chevallier et al., 2006). Indeed, the current study revealed
that the majority of the tested food contact surfaces were
highly contaminated by spoilage flora (Pseudomonas,
Enterobacteriaceae, and yeasts/moulds) (Table 2). Our
results clearly showed that the knives, the tables and the
mincing machines were among the most heavily contami-
nated surfaces within the SSFs (Table 2). In agreement with
other studies, our results also demonstrated the high
variability of the microbial loads on surfaces and equip-
ment in different facilities (Talon et al., 2007). Several
workers have attributed this variability to the microbial
 ARTICLE IN PRESS
320 A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323

100 Batter samples load of the processed food (Lunden et al., 2003b), the
environmental characteristics of the processing facility
80 (temperature, moisture of surfaces) (Chevallier et al.,
2006) and the efficiency of cleaning programs (Reij and
60 Den Aantrekker, 2004); however, the recovery of sub-
stantial numbers (e.g. 45 log CFU/cm2) of Pseudomonas,
40 Enterobacteriaceae, and TVC from the food contact
surfaces of most of the processing facilities, as found in
20 this study, was unexpected (Table 2). This is of particular
concern for the hygienic status of the facilities, since TVC
0 and levels of Enterobacteriaceae are widely used to provide
0-<2 2->4 4->6 6->8 >8 an indication of hygiene and the likelihood of post-
100
1-week sausages processing contamination (Eisel et al., 1997; FSAI, 2001)
as well as the presence of pathogens (FSAI, 2001).
Relative Frequency (%)

80
In this study, S. aureus was recovered from the food
60
contact surfaces of 3 processing facilities at a frequency of
11.8%. It is well known that S. aureus is a principal
40 component of human and animal skin (Borch et al., 1996)
and is frequently isolated from meat-processing facilities,
20 because the microorganism migrates from the carcass
surface to meat during the cutting operation (Borch
0 et al., 1996; Nel et al., 2004; Chevallier et al., 2006).
0-<2 2->4 4->6 6->8 >8 Furthermore, due to the warm and moist environment of
100 meat-processing facilities, proliferation of S. aureus may
Final products occur during processing, especially if cleaning and disin-
80 fection procedures are insufficient (Borch et al., 1996).
Therefore, the distribution of S. aureus within the house-
60 flora of the facilities as found in this study may be the
outcome of cross-contamination of the environment and
40 equipment from raw meat and/or from the food handlers.
However, its distribution may lead to post-process
20 contamination and therefore pose an increased risk in
products that support staphylococcal growth (i.e. products
0 of pH 44.3 or aw40.85; ICMSF, 1996).
0-<2 2->4 4->6 6->8 >8
Salmonella species were recovered from 26.5% of the
Lof CFU/g (Range)
environment and equipment of processing facilities. It has
TVC Pseudomonas Enterococcus been reported that once a production line (e.g. slaughter-
Staphylococci / Kocuria Yeasts & Moulds ing) is contaminated with Salmonella spp. the microorgan-
ism will establish itself on the machinery, equipment and
Enterobacteriaceae Lactic acid bacteria hands of workers (Berends et al., 1997). Indeed, a study in
Fig. 1. Overall distribution (%) of the observed microbial association at
8 slaughtering plants indicated that the prevalence of
different stages during the production of the examined products. Batter presumptive Salmonella spp. was 1.3% (Bacon et al., 2002).
sample: just after stuffing; 1-week sausages: after fermentation; final Moreover, heavily soiled surfaces would be expected to
products: at the time of commercialization. improve the survival of some Salmonella serotypes on such

Table 6
Occurrence of pathogenic bacteria at different stages during the production of the examined products in different facilities

Productsa L. monocytogenes Salmonella spp. S. aureus

Positive samplesb SSFIDc Positive samplesb SSFIDc Positive samplesb SSFIDc

Batter 3/21 (1) 5 (3/3) 1 (3/21) 4 (3/3) 0 (0/21) –

1-week sausage 0/21 (0) – 0 (0/21) – 0 (0/21) –
Final product 9/21 (3) 2 (3/3); 3 (3/3); 5(3/3) 0 (0/21) – 0 (0/21) –
a
Batter: batter sample; 1-week sausage: after fermentation; final product: ready to consumption.
b
Positive samples out of the overall samples tested; value in parenthesis indicate the number of facilities gave positive samples.
c
Small-scale facility identification; number in parenthesis indicate the positive replicates of the facility.
 ARTICLE IN PRESS
A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323
surfaces (De Cesare et al., 2003). Therefore, the markedly
higher results obtained in this study may be explained by
the heavily soiled nature of the food contact surfaces
analyzed.
Consistent with other studies, our results showed that
11.8% of the environmental samples were positive for
L. monocytogenes (Salvat et al., 1995; Tompkin, 2002;
Chevallier et al., 2006). In a previous study, it was shown
that on locations where high aerobic counts were harbored
Enterobacteriaceae were accompanied, and in such loca-
tions L. monocytogenes was also likely to exist (Jessen and
Lammert, 2003). Other studies have related the ability of
this organism to colonize food contact surfaces, in the
presence of Pseudomonas spp. (Sasahara and Zottola,
1993) or Listeria innocua (Bourion and Cerf, 1996);
however, in our study, the presence of L. monocytogenes
could not be correlated only with counts of Enterobacter-
iaceae and Pseudomonas spp., since all sampling sites were
also highly contaminated by spoilage (yeasts-moulds)
or technological (LAB, Staphylococci/Kocuria) flora
(Table 2). Meat processing lines are continuously con-
taminated with L. monocytogenes through incoming raw
products, yet lines that utilize the fermentation process do
not remain persistently contaminating (Lunden et al.,
2003b). This suggests that competing flora may prevent
L. monocytogenes from becoming established on processing
surfaces (Lunden et al., 2003b). Our results indicate that
the competitive flora (LAB) had no destructive effect on
the recovery of pathogens since the presence of
L. monocytogenes was associated with the strong contam-
ination of the processing environment by other flora (i.e.
Pseudomonas, Enterobacteriaceae).
Overall, spoilage (44 log CFU/cm2; Pseudomonas,
Enterobacteriaceae) and pathogenic flora were recovered
from the environment and equipment of almost all SSFs
tested (Table 2; SSF 2, 4–7), with SSFs 4–7 being
characterized as the most heavily contaminated facilities.
Of the 3 facilities (SSF 1, 3, and 5) that operated under
HACCP, 1 (SSF 5) had substantial microbial loads (Table
2) similarly or higher than those that did not have HACCP
in place (SSF 2, 4, 6, and 7). This suggests that in this facility
the HACCP was not applied properly. Moreover according
to food safety and inspection systems, L. monocytogenes is
considered ‘reasonably likely to occur’ especially due to post-
processing contamination and its detection must be addressed
in the HACCP plan (Tompkin, 2002). Since control of
L. monocytogenes is rather achieved through a variety of
prerequisite programs (Tompkin, 2002) that form a solid base
for HACCP (Maunsell and Bolton, 2005) if such programs
are inadequately applied, the presence of L. monocytogenes is
very likely. Therefore, the occurrence of pathogenic and
spoilage flora within the processing surfaces and equipment
as found in this study is more likely indicative of the poor
cleaning and disinfection procedures applied and this may
directly affect the quality and safety of the products.
With regard to product contamination, LAB constituted
the major microflora of the fermented sausages studied
321
(Table 5). Their levels in the final products were similar
to those found in other naturally fermented sausages
(Rebecchi et al., 1998; Samelis et al., 1998; Cocolin et al.,
2001; Lebert et al., 2007). Accordingly, the levels of
Staphylococci/Kocuria in the majority (6/7) of batter
samples were similar to those found in other naturally
fermented sausages (o5 log CFU/g) (Samelis et al., 1998;
Drosinos et al., 2005); however, by the end of ripening
(final products) their population reduced to below the
detection limit (2 log CFU/g) in 5 out of the 7 products.
This elimination reflects their poor competitiveness in the
presence of actively growing aciduric bacteria (Samelis
et al., 1998; Drosinos et al., 2005).
High levels of enterococci were enumerated in 2 of the
end-products tested (Table 5). Several studies have shown
that levels of enterococci increase at early fermentation
stages (4104 CFU/g), while their levels in the end-products
may vary between 102 and 105 CFU/g (Rebecchi et al.,
1998; Samelis et al., 1998; Montel, 2000; Franz et al., 2003).
Moreover, high levels of spoilage flora (Enterobacteriaceae
and Pseudomonas) were enumerated in the majority of the
final products studied (Table 5). In general, Enterobacter-
iaceae and Pseudomonas are usually counted in the first
days of fermentation of both animal and plant origin
foods, while their levels are usually eliminated during
maturation due to the strong competitive effect of LAB on
the rest endogenous flora (Samelis et al., 1998; Spyropou-
lou et al., 2001; Drosinos et al., 2005). However, high levels
of these microorganisms have been reported to survive
during the maturation of fermented sausages (Lebert et al.,
2007), and such levels (4104 CFU/g) indicate product of
low microbiological quality (FSAI, 2001).
Considering the safety of fermented sausages, a rapid pH
drop below 5.3 during fermentation has proved to be
important for the inhibition of Salmonella and S. aureus if
the products are fermented at temperatures above 18 1C
(Lucke, 2000). In the majority of products (6 out of 7) of
the present study, the pH was below 5.3 during fermenta-
tion and the temperature was below 18 1C. Nevertheless,
the low levels of S. aureus and Salmonella found in batter
samples as well as the combination of hurdles during the
fermentation and ripening (aw, pH; Table 6) may have been
able to eliminate both pathogens. Regarding the contam-
ination rate of Listeria, our results (11.8% in batter) were
similar to those of other studies showing the incidence of
L. monocytogenes in almost all sausage preparations
(Samelis et al., 1998; Encinas et al., 1999; Trevenot et al.,
2005). These results are indicative of the incidence of this
pathogen in raw meat and in other materials used for
sausage production (Samelis et al., 1998). Although the
above-mentioned studies have shown that sausage ripening
tends to yield products with or without low levels of
L. monocytogenes, the detection of the microorganism in 3
of the 7 final products raise concerns about possible
product cross-contamination. However, further investiga-
tion is required to analyze higher number of products from
various processing facilities.
 ARTICLE IN PRESS
322 A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323
In conclusion, the current investigation revealed that the
hygienic status of the processing environment and equip-
ment plays an essential role in the microbial quality and
safety of traditional fermented sausages. Indeed, our
results suggest that the incidence of certain pathogens,
such as L. monocytogenes, Salmonella spp. and S. aureus in
food processing plants, may be associated with high
numbers of one or more specific members of the ‘house-
flora’ (Tompkin, 2002; Carpentier and Chassaing, 2004);
however, high levels of ‘house-flora’ are not essentially
indicative of the presence of a certain pathogen. The
insufficient cleaning and disinfection conditions applied in
the majority of the facilities likely contribute to reconta-
mination since high levels of the ‘house-flora’ may
contaminate traditional sausages at any stage of the
processing. According to the microbiological criteria
for foods illustrated by the EU Regulation 2073/2005,
the majority of the products studied were characterized
as unable to support growth of L. monocytogenes (products
with pHp4.4 or awp0.92, products with pHp5.0
and awp0.94) and therefore are of low risk to consumers.
Two products (SSF 2 and 6) marginally supported growth
of L. monocytogenes and might be of higher risk to
consumers; however, in a post-process-contamination
event, the risk associated with the survival of pathogens,
such as L. monocytogenes, is likely to differ among different
fermented and or dry sausages, mainly due to the
complexity and the variety of manufacturing processing
used (Drosinos et al., 2006; Gounadaki et al., 2007;
Skandamis and Nychas, 2007). Therefore, these highly
popular ready-to-eat products may be a source of exposure
of consumers to L. monocytogenes, depending on
their physicochemical characteristics (pH and aw) and
storage conditions (Gounadaki et al., 2007). However,
the level of exposure remains to be evaluated, for until
now contamination levels lower than 103 CFU/g have
never been documented for foods implicated in cases of
invasive listeriosis (FDA/CFSAN, 2003). Apart from
revealing the need for strict control measures within SSFs,
in order to improve the hygiene of the processing
conditions and thereby obtaining finished products of
higher microbiological standards, this study also suggests
motives for further investigation on developing new
disinfection procedures adapted to fermented meat
products.
Acknowledgments
This study has been partly carried out with the financial
support of the Commission of the European Communities,
specific RTD program: Quality of Life and Management of
Living Resources, Key Action 1-Health Food, and
Environment, Project QLK1-CT-2002-02240 (TRADI-
SAUSAGE). It does not necessarily reflect the Commis-
sions views and in no way anticipates its future policy in
this area.
References
Bacon, R.T., Sofos, J.N., Belk, K.E., Hyatt, D.R., Smith, G.C., 2002.
Prevalence and antibiotic susceptibility of Salmonella isolated from
beef animal hides and carcasses. J. Food Prot. 65, 284–290.
Berends, B.R., Van Knapen, F., Snijders, J.M.A., Mossel, D.A.A., 1997.
Identification and quantification of risk factors regarding Salmonella
spp. on pork carcasses. Int. J. Food Microbiol. 36, 199–206.
Borch, E., Nesbakken, T., Christensen, H., 1996. Hazard identification in
swine slaughter with respect to foodborne bacteria. Int. J. Food
Microbiol. 30, 9–25.
Bourion, F., Cerf, O., 1996. Disinfection efficancy against pure-culture
and mixed-population biofilms of Listeria innocua and Pseudmonas
aeroginosa on steel, Teflon and rubber. Sci. Aliments 16, 151–166.
Carpentier, B., Chassaing, D., 2004. Interactions in biofilms between
Listeria monocytogenes and resident microorganisms from food
industry premises. Int. J. Food Microbiol. 97, 111–122.
Chevallier, I., Ammor, S., Laguet, A., Labayle, S., Castanet, V., Dufour,
E., Talon, R., 2006. Microbial ecology of a small-scale facility
producing traditional dry sausage. Food Cont. 17, 446–453.
Cocolin, L., Manzano, M., Cantoni, C., Comi, G., 2001. Denaturing
gradient gel electrophoresis analysis of the 16S rRNA gene V1 region
to monitor dynamic changes in the bacterial population during
fermentation of Italian sausages. Appl. Environ. Microbiol. 67,
5113–5121.
Commission of the European Communities, 2004. Regulation (EC) No
852/2004 of the European Parliament and of the Council of 29 April
2004 on the hygiene of foodstuffs. Off. J. Eur. Union L 139. Available
at: /http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:
2004:226:0003:0021:EN:PDFS. Accessed 20 February 2006.
Commission of the European Communities, 2005. Commission regulation
(EC) No 2073/2005 of 15 November 2005 on microbiological criteria
for foodstuffs. Off. J. Eur. Union L338, 1–26. Available at: /http://
eur-lex.europa.eu/LexUriServ/site/en/oj/2005/l_338/
l_33820051222en00010026.pdfS. Accessed 20 February 2006.
De Cesare, A., Sheldon, B.W., Smith, K.S., Jaykus, L.A., 2003. Survival
and persistence of Campylobacter and Salmonella species under
various organic loads on food contact surfaces. J. Food Prot. 66,
1587–1594.
Deza, M.A., Araujo, M., Garrido, M.J., 2005. Inactivation of Escherichia
coli, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylo-
coccus aureus on stainless steel and glass surfaces by neutral
electrolysed water. Lett. Appl. Microbiol. 40, 341–346.
Drosinos, E.H., Mataragas, M., Xiraphi, N., Moschonas, G., Gaitis, F.,
Metaxopoulos, J., 2005. Characterization of the microbial flora
from a traditional Greek fermented sausage. Meat Sci. 69,
307–317.
Drosinos, E.H., Mataragas, M., Veskovic-Moracanin, S., Gasparik-
Reichardt, J., Hadziosmanovic, M., Alagic, D., 2006. Quantifying
nonthermal inactivation of Listeria monocytogenes in European
fermented sausages using bacteriocinogenic lactic acid bacteria or
their bacteriocins: a case study for risk assessment. J. Food Prot. 69,
2648–2663.
Eisel, W.G., Linton, R.H., Muriana, P.M., 1997. A survey of microbial
levels for incoming raw beef, environmental sources, and ground beef
in a red meat processing plant. Food Microbiol. 14, 273–282.
Encinas, J.-P., Sanz, J.J., Garcıa-Lopez, M.L., Otero, A., 1999. Behaviour
of Listeria spp. in naturally contaminated chorizo (Spanish fermented
sausage). Int. J. Food Microbiol. 46, 167–171.
Fonnesbech-Vogel, B., Jørgensen, L.V., Ojeniyi, B., Huss, H.H., Gram,
L., 2001. Diversity of Listeria monocytogenes isolates from cold-
smoked salmon produced in different smokehouses as assessed by
random amplified polymorphic DNA analyses. Int. J. Food Microbiol.
65, 83–92.
Food and Agriculture Organization of the United Nations (FAO) and the
World Health Organization (WHO), 2004. FAO/WHO guidance to
governments on the application of HACCP in small and/or
 ARTICLE IN PRESS
A.S. Gounadaki et al. / Food Microbiology 25 (2008) 313–323
less-developed food businesses. Available at: /ftp://ftp.fao.org/docrep/
fao/009/a0799e/a0799e00.pdfS. Accessed 3 May 2006.
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 monocytogenes
among selected food categories of ready-to-eat foods. Available at:
/http://www.fsis.usda.gov/Science/Risk_Assessments/index.aspS.
Accessed 20 August 2004.
Food Safety Authority of Ireland (FSAI), 2001.Guidelines for the
interpretation of results of microbiological analysis of some ready-
to-eat foods sampled at point of sale. Guidance Note No. 3.
Available at: /http://www.fsai.ie/publications/guidance_notes/gn3.pdfS.
Accessed 15 April 2004.
Frank, J.F., Koffi, R.A., 1990. Surface-adherent growth of Listeria
monocytogenes is associated with increased resistance to surface
sanitizers and heat. J. Food Prot. 53, 550–554.
Franz, C.M.A.P., Stiles, M.E., Schleifer, K.H., Holzapfel, W.H., 2003.
Enterococci in foods—a conundrum for food safety. Int. J. Food
Microbiol. 88, 105–122.
Gill, C.O., McGinnis, J.C., 2004. Microbial conditions of air knives before
and after maintenance at a beef packing plant. Meat Sci. 68, 333–337.
Gounadaki, A.S., Skandamis, P.N., Drosinos, E.H., Nychas, G.-J.E.,
2007. Effect of packaging and storage temperature on the survival of
Listeria monocytogenes inoculated post-processing on sliced salami.
J. Food Prot. 70, 2313–2320.
Holan, J.T., Bird, J., Hall, K.E., 2004. The microbial ecology of high-risk,
chilled food factories; evidence for persistent Listeria spp. and
Escherichia coli strains. J. Appl. Microbiol. 97, 68–77.
ICMSF, 1996. Characteristics of microbial pathogens. In: Roberts, T.A.,
Baird-Parker, A.C., Tompkin, R.B. (Eds.), Microorganisms in Foods,
Vol. 5. Blackie Academic and Professional, London, pp. 302–304.
International Organization for Standardization, ISO 18593, 2004. Micro-
biology of food and animal feeding stuffs—horizontal methods for
sampling techniques from surfaces using contact plates and swabs,
Switzerland.
Jessen, B., Lammert, L., 2003. Biofilm and disinfection in meat processing
plants. Int. Biodet. Biodeg. 51, 265–269.
Lebert, I., Leroy, S., Giammarinaro, P., Lebert, A., Chacornac, J.P.,
Bover-Cid, S., Vidal-carou, M.C., Talon, R., 2007. Diversity of
microorganisms in the environment and dry fermented sausages of
small traditional French processing units. Meat Sci. 76, 112–122.
Leriche, V., Briandet, R., Carpentier, B., 2003. Ecology of mixed biofilms
subjected daily to a chlorinated alkaline solution: spatial distribution
of bacterial species suggests a protective effect of one species to
another. Environ. Microbiol. 5, 64–71.
Lucke, F.K., 2000. Utilization of microbes to process and preserve meat.
Meat Sci. 56, 105–115.
Lunden, J., Autio, T., Markkula, A., Hellstrom, S., Korkeala, H., 2003a.
Adaptive and cross-adaptive responses to persistent and non-persistent
Listeria monocytogenes strains to disinfectants. Int. J. Food Microbiol.
82, 65–272.
Lunden, J., Autio, T., Sjoberg, A.M., Korkeala, H., 2003b. Persistent and
non-persistent Listeria monocytogenes contamination in meat and
poultry processing plants. J. Food Prot. 66, 2062–2069.
Maunsell, B., Bolton, D.J., 2005. Guidelines for Food Safety Management
on Farms. Teagasc—The National Food Centre, EU-RAIN.
Metaxopoulos, J., Kritikos, D., Drosinos, E.H., 2003. Examination of
microbiological parameters relevant to the implementation of GHP
323
and HACCP system in Greek meat industry in the production of
cooked sausages and cooked cured meat products. Food Cont. 14,
323–332.
Montel, M.C., 2000. Fermented meat products. In: Robinson, R.K., Batt,
C.A., Patel, P.D. (Eds.), Encyclopedia of Food Microbiology.
Academic Press, London, pp. 744–753.
Mossel, D.A.A., Weenk, G.H., Morris, G.P., Struijk, C.B., 1998.
Identification, assessment and management of food-related micro-
biological hazards: historical, fundamental and psycho-social essen-
tials. Int. J. Food Microbiol. 39, 19–51.
Murphy, R.Y., Hanson, R.E., Duncan, L.K., Feze, N., Lyon, B.G., 2005.
Considerations for post-lethality treatments to reduce Listeria mono-
cytogenes from fully cooked bologna using ambient and pressurized
steam. Food Microbiol. 22, 359–365.
Nel, S., Lues, J.F.R., Buys, E.M., Venter, P., 2004. The personal and
general hygiene practices in the deboning room of a high throughtput
red meat abattoir. Food Cont. 15, 571–578.
Peccio, A., Autio, T., Korkeala, H., Rosmini, R., Trevisani, M., 2003.
Listeria monocytogenes occurrence and characterization in meat-
producing plants. Lett. Appl. Microbiol. 37, 234–238.
Rebecchi, A., Crivori, S., Sarra, P.G., Cocconcelli, P.S., 1998. Physiolo-
gical and molecular techniques for the study of bacterial community
development in sausage fermentation. J. Appl. Microbiol. 84,
1043–1049.
Reij, M.W., Den Aantrekker, E.D., 2004. Recontamination as a source of
pathogens in processed foods. Int. J. Food Microbiol. 91, 1–11.
Salvat, G., Toquin, M.T., Michel, Y., Colin, P., 1995. Control of Listeria
monocytogenes in the delicatessen industries: the lessons of a listeriosis
outbreak in France. Int. J. Food Microbiol. 25, 75–81.
Samelis, J., Metaxopoulos, J., 1999. Incidence and principal sources of
Listeria spp. and Listeria monocytogenes contamination in
processed meats and a meat processing plant. Food Microbiol. 16,
465–477.
Samelis, J., Metaxopoulos, J., Vlassi, M., Pappa, A., 1998. Stability and
safety of traditional Greek salami—a microbiological ecology study.
Int. J. Food Microbiol. 44, 69–82.
Sasahara, K.C., Zottola, E.A., 1993. Biofilm formation by Listeria
monocytogenes utilizes a primary colonizing microorganism in flowing
systems. J. Food Prot. 56, 1022–1028.
Skandamis, P.N., Nychas, G.J.E., 2007. Pathogens: risks and control. In:
Toldra, F. (Ed.), Handbook of Fermented Meat and Poultry.
Blackwell Publishing, London, pp. 427–454.
Spyropoulou, K.E., Chorianopoulos, N.G., Skandamis, P.N., Nychas,
G.J.E., 2001. Survival of Escherichia coli O157:H7 during the
fermentation of Sanish-style green table olives (conservolea variety)
supplemented with different carbon sources. Int. J. Food Microbiol.
66, 3–11.
Talon, R., Lebert, I., Lebert, A., Leroy, S., Garriga, M., Aymerich, T.,
Drosinos, E.H., Zanardi, E., Ianieri, A., Fraqueza, M.J., Patarata, L.,
Laukova, A., 2007. Traditional fry fermented sausages produced in
small-scale processing units in Mediterranean countries and Slovakia.
1: Microbial ecosystems of processing environments. Meat Sci., in
press, doi:10.1016/j.meatsci.2007.05.006.
Tompkin, R.B., 2002. Control of Listeria monocytogenes in the food
processing environment. J. Food Prot. 65, 709–725.
Trevenot, D., Delignette-Muller, M.L., Christieans, S., Vernozy-Rozand,
C., 2005. Fate of Listeria monocytogenes in experimentally contami-
nated French sausages. Int. J. Food Microbiol. 101, 189–200.