International Journal of Food Microbiology 57 (2000) 33–42

www.elsevier.nl / locate / ijfoodmicro

Competitiveness and bacteriocin production of Enterococci in the

production of Spanish-style dry fermented sausages
a b a,
Raf Callewaert , Martha Hugas , Luc De Vuyst *
a
Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing,
Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
b
`
Institut de Recerca i Tecnologia Agroalimentaries, Centre de Tecnologia de la Carn, Granja Camps i Armet s /n, 17121 Monells,
Spain

Received 29 April 1999; received in revised form 25 August 1999; accepted 15 January 2000

Abstract

Two different Enterococcus strains of nonmeat origin, namely Enterococcus faecium CCM 4231 and E. faecium RZS
C13, were used as starter cultures in sausage fermentation. Both strains produce a bacteriocin inactive against other lactic
acid bacteria but active against Listeria spp. The competitiveness and anti-Listeria activity of both strains was monitored
during sausage fermentation at both laboratory and pilot scale. The Enterococcus strains were partially competitive during
meat fermentation and strongly inhibited the growth of Listeria spp. The competitiveness of Lactobacillus amylovorus DCE
471, also of a nonmeat origin, was tested too. This strain is characterised as a strong acidifier and produces a bacteriocin
active against other Lactobacilli. However, this strain was not competitive in the meat environment. Since no production of
off-flavours was detected, the Enterococci may be suitable for addition to meat as cocultures to improve food safety.
 2000 Elsevier Science B.V. All rights reserved.

Keywords: Bacteriocin; Enterocin; Enterococcus faecium; Lactic acid bacteria; Meat fermentation; Starter culture

1. Introduction Lactobacillus, Carnobacterium, Pediococcus and

Bacteriocins produced by lactic acid bacteria are
antimicrobial peptides or proteins, which can be used
as natural food preservatives to enhance the shelf life
and safety of food products (De Vuyst and Van-
damme, 1994). Examples of the use of Lactococcus,
*Corresponding author. Tel.: 1 32-2-629-3245; fax: 1 32-2-
629-2720.
E-mail address: ldvuyst@vub.ac.be (L.D. Vuyst)
Leuconostoc bacteriocins as natural food additives or
of the bacteriocin-producing strains as starter cul-
tures or protective cocultures for in situ bacteriocin
production are well known (Kim, 1993; Holzapfel et
al., 1995; Stiles, 1996). For instance, it was shown
that a Lactobacillus plantarum strain producing the
plantaricins S and T may control the natural lactic
acid bacterial microflora in Spanish-style green olive
fermentation (Ruiz-Barba et al., 1994). A paired
starter culture system consisting of nisin-resistant

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34 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42
Leuconostoc mesenteroides and nisin-producing Lac-
tococcus lactis strains may control the cabbage
fermentation to produce sauerkraut by retarding
proliferation of naturally present competitors (Breidt
et al., 1995). Bacteriocin-producing cultures of Lac-
tobacillus curvatus (Vogel et al., 1993b), Lactobacil-
lus sakei (Vogel et al., 1993a; Hugas et al., 1995)
and Lb. plantarum (Campanini et al., 1993) may
control the meat microflora and inhibit food-borne
pathogens during the production of fermented meat
products. The application of bacteriocins produced
by Enterococci and of bacteriocin-producing En-
terococcus starters or protective cultures is well
documented in cheese manufacture (Sulzer and
´ et al., 1994;
Busse, 1991; Villani et al., 1993; Farıas
Giraffa et al., 1994, 1995a,b; Torri Tarelli et al.,
1994; Joosten and Nunez, ˜ 1996; Maisnier-Patin et
al., 1996). One of the most interesting properties of
many enterococcal bacteriocins is their very specific
and strong inhibitory activity against Listeria
(Parente and Hill, 1992; Arihara et al., 1993; Villani
et al., 1993; Vlaemynck et al., 1994; Giraffa, 1995;
Maisnier-Patin et al., 1996). The control of Listeria
spp. is a major concern in food safety since this
pathogen can be present in many food products
(Muriana, 1996). Listeria is able to grow or maintain
itself in meat products (Farber and Losos, 1988;
Johnson et al., 1988; Trussel, 1989), including
fermented meat products (Berry et al., 1990; Luch-
ansky et al., 1992; Campanini et al., 1993).
In a food environment Enterococcus spp. are
mostly associated with dairy products and occasion-
ally with meat. However, bacteriocin-producing En-
terococcus strains with strong anti-Listeria activity
have been isolated from numerous, diverse nondairy
environments including silage (Kato et al., 1994),
fermented olives (Franz et al., 1996), and fermented
sausages (Lyon et al., 1995; Aymerich et al., 1996).
The potential use of enterococcal strains, from a
nonmeat origin (e.g. dairy), in a meat environment
has not been studied until now. In contrast, fermen-
tation and preservation of meat with bacteriocin-
producing Pediococcus (Berry et al., 1990; Sabel et
al., 1991; Foegeding et al., 1992; Luchansky et al.,
1992) and Lactobacillus (Schillinger et al., 1991;
Campanini et al., 1993; Winkowski et al., 1993;
Vogel et al., 1993a,b; Hugas et al., 1995) has already
been described (Stiles and Hastings, 1991; McMul-
len and Stiles, 1996; Muriana, 1996). Recently, a
bacteriocin-producing transconjugant Lc. lactis
Cheddar cheese starter was found suitable to use as a
meat starter culture (Coffey et al., 1998).
In this paper, the competitiveness of two bac-
teriocinogenic Enterococcus faecium strains and one
bacteriocinogenic Lactobacillus amylovorus strain
from different nonmeat origin is compared in
Spanish-style dry sausage fermentation. The bac-
teriocin production by the Enterococcus strains was
monitored against Listeria spp. during sausage fer-
mentation at both laboratory and pilot scale.
2. Materials and methods
2.1. Bacterial strains and media
The bacteriocin-producing (Bac 1 ) strains E.
faecium RZS C13 (isolated from cheese; Vlaemynck
et al., 1994), E. faecium CCM 4231 (isolated from
calf rumen; Laukova´ et al., 1993) and Lb.
amylovorus DCE 471 (isolated from corn steep
liquor; De Vuyst et al., 1996) were tested for their
competitiveness in Spanish-style dry sausage fermen-
tation. Lb. curvatus CTC 371 (Bac 2) and Lb. sakei
CTC 494 (Bac 1 ) were used as negative and positive
control for bacteriocin production, respectively. The
bacteriocin production by E. faecium RZS C13, E.
faecium CCM 4231 and Lb. sakei CTC 494 was
monitored by the use of sensitive, nonpathogenic
variants of Listeria, namely Listeria innocua CTC
1012, List. innocua CTC 1014 and Listeria ivanovii
LTH 3097, respectively.
All strains were stored at 2 808C in the appro-
priate cultivation broth containing 50% (v / v) gly-
cerol (Merck, Darmstadt, Germany). Lactic acid
bacteria were cultivated in de Man–Rogosa–Sharpe
(MRS) broth (Difco, MI, USA) and Listeria spp.
were cultivated in tryptic soy broth (TSB) (Difco)
supplemented with 0.6% yeast extract (Difco). Pal-
cam selective medium (Merck) was used to enumer-
ate the Listeria density of the samples. Solid media
and overlay agars were prepared by adding 1.5%
(w / v) or 0.7% (w / v) agar (Difco), respectively, to
the appropriate broth.
2.2. Inoculum preparation
To prepare the inoculum, lactic acid bacteria and
Listeria spp. were cultivated twice overnight at 308C
in MRS or TSB broth, respectively, starting from a
 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42
culture stored at 2 808C. Subsequently, 10 ml of an
overnight culture were centrifuged (12 000 g, 10
min). Each of the pelleted cultures was resuspended
in 1 ml of fresh cultivation broth, supplemented with
1 ml of glycerol and stored until required at 2 408C.
Lactic acid bacteria and Listeria populations were
enumerated on MRS or TSB agar plates, respective-
ly, and expressed as colony forming units (CFU) per
ml. Approximately 10 6 CFU of the starter culture
and 10 4 CFU of the Listeria indicator culture per
gram of meat batter were used as inocula in the meat
fermentation experiments. Either one Listeria in-
dicator strain or a mixed culture of three Listeria
indicator strains in a proportion of 1:1:1 were used
(see below).
2.3. Sausage manufacturing
The meat batter used for the preparations of
Spanish-style dry fermented sausages contained the
following ingredients (g / kg): lean pork (800), back
fat pork (200), sodium chloride (25.0), sodium nitrite
(0.1), potassium nitrate (0.3), sodium ascorbate
(0.5), sodium pyrophosphate pH 5.0 (1.5), dextrose
(7.0), lactose (10.0), skimmed milk powder (10.0),
sodium caseinate (10.0), Ponceau 4R (0.02) SKW
Biosystems, Rubı, ´ Barcelona, Spain, black pepper
(3.0) and water (50). The meat was tempered at 2 1
to 08C.
In a first series of experiments, sausage fermen-
tation was followed in a model sausage system
(Hugas et al., 1995). Five experiments were set up to
test the five different starter cultures mentioned
above. Each experiment was done in triplicate.
Therefore, the meat batter was prepared by mixing
the different ingredients manually. Subsequently, a
mixture of indicator strains (List. innocua CTC 1012,
List. innocua CTC 1014 and List. ivanovii LTH
3097) was added, followed by the starter culture (E.
faecium CCM 4231, E. faecium RZS C13, Lb. sakei
CTC 494 or Lb. curvatus CTC 371). For the trials
with Lb. amylovorus DCE 471 as starter culture, no
Listeria indicator culture was added. Petri dishes
were used as casings. Each casing was filled with
approximately 100 g of meat batter. The plates were
incubated at 228C. After 3 days, when the pH had
dropped to a value of 5.2 to 5.0 (see Sausage
sampling and analysis), the plates were stored at
158C to slow down the fermentation.
In a second series of experiments, real sausages
35
were made in a pilot plant (Institut de Recerca i
`
Tecnologia Agroalimentaries, Monells, Spain). In
these experiments E. faecium CCM 4231, E. faecium
RZS C13 or Lb. sakei CTC 494 were used as starter
culture. List. innocua CTC 1014 was used as in-
dicator strain. The meat batter mentioned above was
prepared in a vacuum mixer. Approximately 500 g of
the mixture was stuffed into 7 cm diameter collagen
casings and the whole was finally dipped into a
solution of Penicillium nalgiovense. All trials were
done in triplicate. The fermentation was performed at
a temperature of 22 to 248C, and at a relative
humidity of 90 to 95%. When the pH had dropped to
a value of 5.2 to 5.0 (see Sausage sampling and
analysis), the sausages were dried at a temperature of
158C and at a relative humidity of 70 to 80% during
4 weeks.
2.4. Sausage sampling and analysis
Model and real sausages were sampled at regular
time intervals. To follow the fermentation profile, the
pH was measured by inserting a pH electrode
(Ingold A4-S7-35; Crison, Switzerland) into the
meat. Bacteria counts were determined as follows.
Ten grams of meat were taken aseptically from the
centre of the model or real sausages and diluted with
90 ml of sterile saline solution (0.85% sodium
chloride; Merck) enriched with bacteriological pep-
tone (1 g / l; Difco). The sample was homogenised
for 1 min in a Stomacher Labblender (model 400;
Cooke Laboratory Products, Alexandria, VA, USA).
The total lactic acid bacteria counts were determined
on MRS agar. Serial tenfold dilutions were plated.
The plates were incubated anaerobically at 308C for
48 h. Strain domination of the starter culture was
verified by testing at least four randomly chosen
colonies according to a progressive colony sampling
plan based on the accumulated binomial distribution
(Garriga et al., 1996). The authenticity of bacterial
colonies was identified by plasmid profile analysis
according to the method of Anderson and McKay
(1983). Their ability to produce bacteriocins and
inhibit a sensitive indicator strain was tested by
direct antagonism (Barefoot and Klaenhammer,
1983). Bacteriocin production in the meat was
monitored by the reduction of the cell count (CFU)
of sensitive Listeria indicator strains. Listeria spp.
were enumerated on selective Palcam agar (Merck).
Plates were incubated aerobically at 308C for 48 h.
36 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42

Bacteria counts and pH values of three parallel

experiments were averaged and standard deviations
were calculated.

2.5. Sensory evaluation

The sausages manufactured with the Enterococcus

strains as starter cultures and not inoculated with
Listeria were evaluated by a panel of ten members.
The results were compared with uninoculated natu-
rally fermented sausages. Slices of dry fermented
sausages (3 mm thick) were checked for appearance,
texture and flavour after rinsing the mouth with
water. The following parameters were evaluated:
redness, hardness, colour, sweetness, saltiness, acid
taste, bitterness, sodium glutamate flavour, musti-
ness, cohesiveness, fat melting, uniformity of colour,
and crustiness. Results of the appreciation were
scaled from 0 (low intensity or absence) to 10 (high
intensity). The data were statistically treated using
the general linear model programme from the statisti-
cal package SAS (SAS, 1985).

3. Results

The competitiveness of the bacteriocin-producing

strains Lb. amylovorus DCE 471, E. faecium CCM
4231 and E. faecium RZS C13 was tested in
Spanish-style dry sausage fermentation. Both En-
terococcus strains produced a strong anti-Listeria
bacteriocin while the Lactobacillus strain produced a
bacteriocin only active against Lactobacillus spp.
and not against Listeria spp.

3.1. Competitiveness of E. faecium during sausage

fermentation
The sausage fermentation process was first simu-
lated in a model sausage system. Five different
strains were separately used as starter culture: Lb.
amylovorus DCE 471, E. faecium CCM 4231 and E.
faecium RZS C13 were tested as new starter cultures
and compared to Lb. sakei CTC 494 and Lb.
curvatus CTC 371, two strains able to dominate the
sausage fermentation process (Hugas et al., 1995).
The pH of the dry fermented sausages was monitored
to follow the fermentation profiles. Acidification
took place mainly during the first 3 days (Fig. 1a).
Fig. 1. Model sausage fermentation: (a) pH profile, (b) total lactic
acid bacteria count on MRS agar and (c) Listeria count on Palcam
agar of the experimental sausage trial inoculated with Lactobacil-
lus curvatus CTC 371 (j), Lactobacillus sakei CTC 494 (♦),
Lactobacillus amylovorus DCE 471 (s), Enterococcus faecium
RZS C13 (h) and Enterococcus faecium CCM 4231 (쏻) as starter
culture. The values are the average of three experiments. The error
bars represent the standard deviations.
Each of the strains acidified the model sausage to the
same extent. The lactic acid bacteria growth profiles
were similar for the different experiments (Fig. 1b).
 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42 37

The meat, which was naturally contaminated with
approximately 1.0 ? 10 4 CFU / g of lactic acid bac-
teria, was inoculated with approximately 1.0 ? 10 6
CFU / g of starter bacteria. After 3 days of fermen-
tation the total lactic acid bacteria count averaged
3.0 ? 10 8 CFU / g. Only the fermentation with Lb.
sakei CTC 494 as starter culture reached a higher
cell count of 1.5 ? 10 9 CFU / g. Plasmid profiles of
bacterial colonies isolated from the Lb. sakei CTC
494 and the Lb. curvatus CTC 371 fermentation
experiments confirmed the competitiveness of these
strains in a meat environment: identical profiles were
found for at least four randomly chosen colonies. In
contrast, in the three other experiments a variation of
plasmid profiles was observed. After 3 and 7 days,
approximately 50% of the colonies isolated from the
two Enterococcus starter culture experiments dis-
played bacteriocin activity and possessed the same
colony morphology and plasmid profile as compared
to the initial starter culture. This indicates that these
Enterococcus starters were only partially competi-
tive. Plasmid profiles of bacteriocin-producing
strains isolated from the Enterococcus trials are
shown in Fig. 2. After 3 days, Lb. amylovorus DCE
471 could not be recovered from the meat at all. No
amylovorin L471-producing colonies could be iso-
lated. Colony morphology and plasmid profiles of
the colonies isolated, showed the presence of a
heterogeneous lactic acid bacterium population. It
turned out that the indigenous microflora had over-
grown the Lb. amylovorus starter culture.
For the real sausage fermentation, where Lb. sakei
CTC 494, E. faecium CCM 4231 and E. faecium
RZS C13 were used as starter cultures, identical
results as for the model sausage fermentation were
obtained. Fermentation of real sausage occurred at
similar acidification rates (Fig. 3a). The total lactic
acid bacteria counts were similar for both Enterococ-
cus trials. The total lactic acid bacteria count for the
Lb. sakei CTC 494 trial was more than 1 log CFU / g
higher as compared to the Enterococcus trials (Fig.
3b). Whereas Lb. sakei CTC 494 was dominating the
fermentation, both Enterococcus strains were again
only partially competitive in the meat environment.
3.2. Competitiveness of E. faecium during the
sausage drying and maturation process
During the sausage fermentation E. faecium
strains were able to maintain 50% of the total lactic
acid bacteria population (see above). Upon further
ripening, the Enterococcus starters gradually dis-
appeared from the microflora. After 21 days, no
colony could be isolated and identified as being the

Fig. 2. Plasmid profiles of Lactobacillus sakei CTC 494 (lane 6), Enterococcus faecium RZS C13 (lane 11) and Enterococcus faecium
CCM 4231 (lane 16). Lanes 1 to 5: plasmid profiles of colonies isolated from a noninoculated sausage trial. Lanes 7 to 10, lanes 12 to 15
and lanes 17 to 20: plasmid profiles of colonies isolated from the sausage trials inoculated with the Lb. sakei CTC 494 starter, E. faecium
RZS C13 and E. faecium CCM 4231, respectively.
38 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42
Fig. 3. Real sausage fermentation: (a) pH profile, (b) total lactic
acid bacteria count on MRS agar and (c) Listeria count on Palcam
agar of the experimental sausage trial inoculated with Lactobacil-
lus sakei CTC 494 (♦), Enterococcus faecium RZS C13 (h) and
Enterococcus faecium CCM 4231 (쏻) as starter culture. The
values are the average of three experiments. The error bars
represent the standard deviations.
original bacteriocin-producing Enterococcus starter
culture. However, total lactic acid bacteria counts
remained constant (Fig. 3b). This indicates that
bacteriocin-producing enterococcal meat starter cul-
tures may be very effective at early stages of meat
fermentation guaranteeing the production of micro-
biologically safe end products.
3.3. Bacteriocin production and inhibition of
Listeria
Bacteriocin production by the starter cultures E.
faecium RZS C13, E. faecium CCM 4231 and Lb.
sakei CTC 494 in the model sausage was monitored
by the reduction of the Listeria count of samples
plated on Palcam agar. The results were compared to
Listeria growth inhibition by the nonbac-
teriocinogenic Lb. curvatus CTC 371 strain. Due to
the drop in pH, the bacteriocin-negative control was
able to decrease the Listeria count by 0.86 log
CFU / g (Fig. 1c). The sakacin K-producing Lb. sakei
CTC 494 strain decreased the Listeria count by 1.65
log CFU / g. Both Enterococcus strains inhibited
Listeria growth to a greater extent than this positive
control. The Listeria count decreased by 2.42 log
CFU / g and 1.87 log CFU / g for the E. faecium RZS
C13 and the E. faecium CCM 4231 strains, respec-
tively (Fig. 1c). Similar results were obtained during
real sausage fermentation (Fig. 3c). The Enterococ-
cus strains were able to reduce the Listeria count by
2.00 log CFU / g after 3 days, compared to a decrease
of 1.26 log CFU / g for the Lb. sakei CTC 494 strain.
The Listeria count remained low during the follow-
ing maturation process and was nearly two-times
lower in the Enterococcus starter culture trials as
compared to the trials with the Lb. sakei CTC 494
strain. A total decrease of 3.28 and 3.25 log CFU / g
was obtained after 28 days for the E. faecium RZS
C13 and the E. faecium CCM 4231 strains, respec-
tively.
3.4. Organoleptic analysis
Sausages for organoleptic analysis were made as
described above but omitting the Listeria indicator.
These sausages were presented to a taste panel.
Sausage fermentation with E. faecium RZS C13 or
E. faecium CCM 4231 as starter culture were
compared to sausage fermentations performed with-
out these starter cultures. Both sausage types gave
similar results (Table 1). Fermentations with the
enterococcal starters resulted in a sweeter and less
acidic taste compared to a noninoculated sausage.
 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42 39

Table 1 C13. Both enterococcal strains produce a strong

Sensorial analysis (least-squares means for different characteris- anti-Listeria bacteriocin designated as enterocin
tics) and comparison of sausages manufactured with or without
Enterococcus faecium strains as starter cultures a 4231 (Laukova´ et al., 1993) and enterocin 13
(Vlaemynck et al., 1994), respectively. Lb.
Attribute Lots
amylovorus DCE 471 is a strong acid producer and
E. faecium E. faecium Without produces the bacteriocin amylovorin L471, which is
RZS C13 CCM 4231 inoculation characterised by a narrow spectrum inhibiting only
Colour 5.91 5.50 5.65 some Lactobacilli (De Vuyst et al., 1996). This strain
Uniform colour 6.00 5.56 5.75 was not able to lead the sausage fermentation since it
Crustiness 4.20 3.94 3.90
was immediately overgrown by the indigenous mi-
Sweetness 1.40 b 1.22 b 0.75 c
Saltiness 3.28 3.39 3.45 croflora. Sausage fermentation occurred at 228C to
Acid taste 2.90 c 3.42 c 4.53 b 248C, which does not favour growth of this Lac-
Bitterness 1.10 1.33 1.25 tobacillus strain since it grows optimally at higher
Glutamate 1.75 1.67 1.80 temperatures up to 458C (De Vuyst et al., 1996). The
Mustiness 4.55 b 5.17 b 2.00 c
E. faecium strains are less strong acidifiers but are
Hardness 6.15 6.44 6.15
Cohesiveness 6.15 c 6.33 c 4.05 b able to grow at lower temperatures. In this study,
Fat melting 2.00 1.22 1.80 both were shown to be partially competitive and able
a
The appreciation was scaled from 0, low intensity or absence, to maintain themselves among the meat microflora
to 10, high intensity. during fermentation. However, upon further matura-
b
and c , means within the same row with different superscript tion they disappeared from the food matrix. This
differ (P , 0.05). indicates their potential usefulness as coculture to
control the early stages of meat fermentation.
These properties were highly appreciated by the Compared to the bacteriocinogenic Lb. sakei CTC
consumer. The only less appreciated property was a 494 strain that was used as a positive control,
musty flavour. Listeria inhibition was much stronger when E.
faecium CCM 4231 or E. faecium RZS C13 were
used as starter culture. The bacteriocins from both
Enterococcus strains are indeed strongly active
4. Discussion against Listeria and do not influence growth of either
starter or nonstarter lactic acid bacteria, a typical
In contrast with fermentation of sterile milk, meat feature of many bacteriocins from Enterococcus spp.
fermentations do not start from a sterile raw material. (Parente and Hill, 1992; Villani et al., 1993; Giraffa,
Production of dry fermented sausage starts from 1995). The Enterococci investigated here demon-
nonprocessed raw meat. Food spoilage microorga- strate very promising characteristics with regard to
nisms or pathogens such as Listeria can easily their application, combined with other lactic starter
contaminate the meat. Fast growth of highly com- cultures, to improve food safety, in particular con-
petitive starter cultures and acidification of the meat cerning Listeria. A bacteriocin-producing E. faecium
are essential to the prevention of spoilage and strain was also proposed as an adjunct culture in the
pathogenic bacterial growth. The use of strains that production of cheese to prevent Listeria growth in
produce antimicrobial proteins or bacteriocins could milk (Giraffa et al., 1995a,b). The enhanced Listeria
be a major advantage in this competition. It has inhibition by the Enterococcus strains used in this
indeed been shown that dominance of Lb. curvatus work seems to be particularly interesting in meat
LTH 1174 (Vogel et al., 1993b) and Lb. sakei CTC fermentations. The suppression of Listeria during
494 (Hugas et al., 1995) in dry sausage fermentation sausage maturation suggests the continued presence
was attributed to bacteriocin production. and activity of enterocin throughout the production
In this paper, three strains of nonmeat origin were process. As a consequence, the growth of further
tested for their competitiveness in Spanish-style dry contaminants may also be prevented using enterocin-
fermented sausage production: Lb. amylovorus DCE producing cocultures. Similar characteristics were
471, E. faecium CCM 4231 and E. faecium RZS described for a pediocin-producing Pediococcus
40 R. Callewaert et al. / International Journal of Food Microbiology 57 (2000) 33 – 42
acidilactici strain in turkey sausages (Luchansky et
al., 1992).
The in situ action of many bacteriocins in a meat
environment is unsatisfactory. This may be ascribed
to limited bioavailable concentrations due to a poor
solubility (as a result of an unfavourable pH), limited
diffusion and localised accumulation (as a result of
binding to food particles and surfaces). Additional
factors responsible for low in situ bacteriocin activity
include decreased rates of bacteriocin production
and / or environmental factors including the presence
of inhibitors, proteases and hydrophobic interactions
with fats and proteins (Hurst, 1981; Jung et al., 1992;
Daeschel, 1993). For instance, the low carbohydrate
content and low ripening temperature of fermented
meat products may be responsible for a low bac-
teriocinogenic in situ activity (Vogel et al., 1993b;
Hugas et al., 1995). Furthermore, Listeria inhibition
in dry fermented sausages by bacteriocinogenic lactic
starter cultures is strongly dependent on the meat
formulation used (Hugas et al., 1996) and the starter
culture population density applied (Buncic et al.,
1997). The occurrence of resistance of Listeria
towards bacteriocins is a concern for the use of these
anti-Listeria proteins (Harris et al., 1991; Davies and
Adams, 1994; Rekhif et al., 1994). The application
of a bacteriocin mixture may avoid development of
resistant Listeria (Hanlin et al., 1993; Davies and
Adams, 1994; Rekhif et al., 1994). There is hence a
requirement for additional or better-adapted meat
starter cultures or protective cultures to improve the
food safety of naturally fermented meat products.
Enterococcus strains as those used in this work are
very promising. In addition, sausages fermented with
E. faecium RZS C13 and E. faecium CCM 4231
were sweeter and less acidic than traditionally pre-
pared dry fermented sausages. These properties are
highly appreciated by the consumers. Sausages pro-
duced with the dairy starter culture Lc. lactis had
also a milder flavour than sausages produced with
Lb. sakei and Staphylococcus carnosus as starter
cultures (Coffey et al., 1998).
In this paper, it was shown that the Enterococcus
strains, E. faecium CCM 4231 and E. faecium RZS
C13, were partially competitive during meat fermen-
tation, strongly inhibited the growth of Listeria, and
improved the organoleptic properties of mature dry
fermented sausages. Such characteristics identify
these strains of E. faecium as suitable cultures for
addition to starter cultures applied to dry fermented
sausages.
Acknowledgements
Part of this research was financially supported by
the Research Council of the Vrije Universiteit Brussel
(VUB), the Institut de Recerca i Tecnologia Ag-
`
roalimentaries (IRTA), and the Biotechnology and
FAIR Programs of the European Commission (grants
BIO2-CT943055, ERB-CIPACT-940160 and FAIR-
CT97-3227). R. Callewaert received a travel grant of
the Fund for Scientific Research—Flanders (FWO—
Flanders). The authors thank Dr. ir. Geertrui Vla-
emynck and Dr. Andrea Laukova´ for providing the
Enterococcus strains used in this study. The authors
also gratefully acknowledge the technical assistance
of Mr. Luis Guerrero in the sensory evaluation.
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