1-s2.0-S016816050500406X-main

International Journal of Food Microbiology 106 (2006) 1 – 24
Review
The role and application of enterococci in food and health
M.R. Foulquié Moreno a,1 , P. Sarantinopoulos b,1,2 , E. Tsakalidou b , L. De Vuyst a, *
a Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing (IMDO), Department of Applied Biological Sciences and
Engineering, Vrije Universiteit Brussel (VUB), Pleinlaan 2, B-1050 Brussels, Belgium
b Laboratory of Dairy Research, Department of Food Science and Technology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece
Received 19 February 2005; accepted 5 June 2005
www.elsevier.com/locate/ijfoodmicro
Abstract
The genus Enterococcus is the most controversial group of lactic acid bacteria. Studies on the microbiota of many traditional cheeses in the
Mediterranean countries have indicated that enterococci play an important role in the ripening of these cheeses, probably through proteolysis,
lipolysis, and citrate breakdown, hence contributing to their typical taste and flavour. Enterococci are also present in other fermented foods, such
as sausages and olives. However, their role in these products has not been fully elucidated. Furthermore, the production of bacteriocins by
enterococci is well documented. Moreover, enterococci are nowadays used as probiotics. At the same time, however, enterococci have been
associated with a number of human infections. Several virulence factors have been described and the number of vancomycin-resistant
enterococci is increasing. The controversial nature of enterococci has prompted an enormous increase in scientific papers and reviews in recent
years, where researchers have been divided into two groups, namely pro and contra enterococci. To the authors’ impression, the negative traits
have been focused on very extensively. The aim of the present review is to give a balanced overview of both beneficial and virulence features of
this divisive group of microorganisms, because it is only acquaintance with both sides that may allow their safe exploitation as starter cultures or
co-cultures.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Enterococcus; Food; Health; Functional properties; Pathogenesis
Contents
1. The genus Enterococcus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2. Functional properties of enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.1. Bacteriocin production by enterococci. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2.2. Citrate metabolism by enterococci. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.3. Proteolysis and lipolysis by enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3.1. Proteolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.3.2. Lipolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.4. Enterococci as probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
3. Pathogenesis of enterococci. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1. Cytolytic activity of enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2. Antibiotic resistance of enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.3. Virulence factors of enterococci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
* Corresponding author. Tel.: +32 2 6293245; fax: +32 2 6292720.
E-mail address: ldvuyst@vub.ac.be (L. De Vuyst).
1 M.R. Foulquié Moreno and P. Sarantinopoulos have equally contributed to this manuscript.
2 Current address: DSM Food Specialties, Research and Development, Process Technology Department, P.O. Box 1, 2600 MA, Delft, The Netherlands.
0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2005.06.026

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M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1 – 24
3.3.1. Aggregation substance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.3.2. Gelatinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.3.3. Extracellular surface protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.4. Opsonophagocytic assay to assess the safety of potential enterococcal pathogens . . . . . . . . . . . . . . . . . . . . . . . 10
4. Enterococci in food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.1. Cheese products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.1.1. Presence of enterococci in cheese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.1.2. Applicability of enterococci as starter or adjunct cultures in cheese . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.1.3. Applicability of bacteriocinogenic enterococci and/or their enterocins in cheese . . . . . . . . . . . . . . . . . . . 12
4.2. Meat products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2.1. Presence of enterococci in meat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.2.2. Applicability of bacteriocinogenic enterococci and/or their enterocins in meat products . . . . . . . . . . . . . . . 15
4.3. Vegetables and olives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1. The genus Enterococcus
The genus Enterococcus belongs to a group of microorganisms
known as lactic acid bacteria (LAB). Enterococci
are Gram-positive, non-sporeforming, catalase-negative, oxidase-negative,
facultative anaerobic cocci that occur singly,
in pairs, or in chains. From a taxonomic point of view, the
genus Enterococcus has been reviewed several times
(Schleifer and Kilpper-Bälz, 1987; Stackebrandt and Teuber,
1988; Devriese and Pot, 1995; Hardie and Whiley, 1997). In
1984, DNA–DNA hybridisation and 16S rRNA sequencing
studies indicated that the species Streptococcus faecium and
S. faecalis were sufficiently distinct from other streptococci,
and Schleifer and Kilpper-Bälz (1984) proposed their
transfer to the genus Enterococcus. To date, 28 species,
namely E. asini, E. avium, E. canis, E. casseliflavus, E.
cecorum, E. columbae, E. dispar, E. durans, E. faecalis, E.
faecium, E. flavescens, E. gallinarum, E. gilvus, E.
haemoperoxidus, E. hirae, E. malodoratus, E. moraviensis,
E. mundtii, E. pallens, E. phoeniculicola, E. pseudoavium,
E. raffinosus, E. ratti, E. saccharolyticus, E. saccharominimus,
E. solitarius, E. sulfureus, and E. villorum, have
been added to the genus on the basis of phylogenetic
evidence strengthened by 16S rRNA DNA sequencing and/
or DNA–DNA hybridization studies. However, some reclassifications
may occur in the near future. For instance, E.
flavescens and E. casseliflavus have insufficient differences
to be classified separately (Devriese and Pot, 1995;
Descheemaeker et al., 1997), and E. avillorum is an earlier
heterotypic synonym of E. porcinus (De Graef et al., 2003).
Enterococci grow optimally at a temperature of 35 -C,
although most species in the genus will grow at temperatures
ranging from 10 to 45 -C. They can also grow in the
presence of 6.5% NaCl, at pH 9.6, and they survive heating
at 60 -C for 30 min. Exceptions include E. dispar and E.
sulfureus (Martínez-Murcia and Collins, 1991), E. malodoratus
(Collins et al., 1984) and E. moraviensis (Svec et al.,
2001), which do not grow at 45 -C, and E. cecorum and E.
columbae, which do not grow at 10 -C (Devriese et al.,
1993). E. avium, E. saccharominimus, E. cecorum and E.
columbae grow poorly or not at all in the presence of 6.5%
NaCl (Devriese et al., 1990, 1993; Vancanneyt et al., 2004).
Most enterococcal species can hydrolyse aesculin in the
presence of 40% bile salts. Enterococcal species possess the
Lancefield group D antigen with the exception of E.
cecorum, E. columbae, E. dispar, E. pseudoavium, E.
saccharolyticus and E. sulfureus (Hardie and Whiley,
1997). E. mundtii and E. casseliflavus are yellow-pigmented,
and E. gallinarum and E. casseliflavus are motile (Schleifer
and Kilpper-Bälz, 1987).
2. Functional properties of enterococci
Studies on the microbiota of traditional cheeses in the
Mediterranean countries, which are mainly produced from
raw ewes’ or goats’ milk, and less commonly from cows’
milk, have indicated that enterococci play an important role
in the ripening of these cheeses, probably through proteolysis,
lipolysis, and citrate breakdown, hence contributing to
their typical taste and flavour, (Coppola et al., 1990;
Litopoulou-Tzanetaki and Tzanetakis, 1992; Ledda et al.,
1994; Giraffa et al., 1997; Manolopoulou et al., 2003). They
are also present in other fermented food products, such as
sausages (Franz et al., 1999a, 2003; Giraffa, 2002; Hugas et
al., 2003) and olives (Fernández-Diaz, 1983; Franz et al.,
1996, 1999a; Floriano et al., 1998; Ben Omar et al., 2004).
Enterococci have the ability to produce bacteriocins, the socalled
enterocins, which are small peptides with antimicrobial
activity towards closely related Gram-positive bacteria
including spoilage or pathogenic bacteria, such as Listeria
(De Vuyst and Vandamme, 1994). Moreover, enterococci are
used in some countries as probiotics (Franz et al., 1999a,
2003).
2.1. Bacteriocin production by enterococci
The bacteriocins produced by LAB belong to the large
group of ribosomally synthesised, small, cationic, amphiphilic
(rather hydrophobic), antimicrobial peptides, naturally produced
by microorganisms, which vary in spectrum and mode of

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 3
activity, molecular structure and molecular mass, thermostability,
pH range of activity, and genetic determinants (Klaenhammer,
1993; De Vuyst and Vandamme, 1994; Nes et al.,
1996; Nissen-Meyer and Nes, 1997; Moll et al., 1999; Ennahar
et al., 2000; Cleveland et al., 2001; McAuliffe et al., 2001;
Riley and Wertz, 2002).
Based on structural, physicochemical and molecular properties,
bacteriocins from LAB can be subdivided into three
major classes (Klaenhammer, 1993; Nes et al., 1996).
Nonetheless, this classification is continuously being reviewed
and it is evolving with the accumulation of knowledge and the
appearance of new bacteriocins (Cleveland et al., 2001;
Ennahar et al., 2000; Riley and Wertz, 2002; Rodríguez et
al., 2003). Class I bacteriocins are lantibiotics, i.e. small,
cationic, hydrophobic, and heat-stable peptides that contain
unusual amino acids (e.g. the thioether amino acids lanthionine
and/or 3-methyl-lanthionine) that are post-translationally
formed. Class II bacteriocins are small, cationic, hydrophobic,
heat-stable peptides that are not post-translationally modified,
except for cleavage of a leader peptide from the prebacteriocin
peptide. Within this class, three subclasses can be distinguished:
subclass IIa or pediocin-like bacteriocins with a strong
antilisterial effect, possessing the consensus sequence YGNGV
in their N-terminus; subclass IIb or bacteriocins that require
two polypeptide chains for full activity; and subclass IIc or
bacteriocins that do not belong to the other subgroups. Class III
bacteriocins are a group of large, hydrophilic, heat-labile
proteins.
Since 1955, when the first bacteriocin-like substance within
the group D streptococci was reported (Kjems, 1955), a large
number of enterocins has been studied. Krämer and Brandis
(1975) reported the anti-Listeria activity of the enterocin E1A
produced by E. faecium E1. Nowadays, the ability of
enterococci to inhibit Listeria spp. is well known, which may
be explained by the close phylogenetic relationship of
enterococci and listeriae (Stackebrandt and Teuber, 1988;
Devriese and Pot, 1995). The first enterocin purified to
homogeneity was the enterocin AS-48 produced by E. faecalis
S-48 (Gálvez et al., 1989; Martínez-Bueno et al., 1994), which
was defined as a cyclic peptide antibiotic. Since then, the
number of new enterocins characterised is increasing. However,
many of these molecules have not been purified to
homogeneity. This is the case for the bacteriocins produced
by E. faecalis E-1 (Bottone et al., 1971), E. faecium E1
(Krämer and Brandis, 1975), E. faecalis E23 (Nakagawa,
1979), E. faecium (Harasawa et al., 1980), E. faecium S-34
(Nakagawa and Matsuo, 1981), E. faecium 3(Krämer et al.,
1983), E. faecium 25 (Reichelt et al., 1984), E. faecalis K4
(Kühnen et al., 1985), E. faecalis DS16 (Ike et al., 1990), E.
faecium 100 (Kato et al., 1993), E. faecalis 226 NWC (Villani
et al., 1993), E. faecium NA01 (Olasupo et al., 1994), E.
faecium 7C5 (Torri Tarelli et al., 1994), and E. faecium L1
(Lyon et al., 1995).
Enterocins are found within the class I, class IIa, class IIc,
and class III bacteriocins mentioned above (Table 1). Other
enterocins that at first were considered as new enterocins, but
were actually identical to existing ones, are listed in Table 2. In
addition, the bacteriocins produced by E. faecalis strains are
referred to as:
Type 1: cytolysin (bacteriocin/hemolysin) from E. faecalis
DS16 (Gilmore et al., 1994). This two-component lantibiotic
displays both hemolytic and bacteriocin activity.
Type 2: the cyclic peptide antibiotic AS-48 (enterocin AS-
48) from E. faecalis S-48 (Martínez-Bueno et al., 1994).
This compound is active towards both Gram-positive and
Gram-negative bacteria. In contrast, the identical enterocin 4
produced by E. faecalis INIA 4 is only active towards
Gram-positive bacteria (Joosten et al., 1996).
Type 3: bacteriocin 31 from E. faecalis YI17 (Tomita et al.,
1996) with a narrow antibacterial spectrum.
Type 4: enterocin 1071A and enterocin 1071B from E.
faecalis BEF 1071 (Balla et al., 2000). These enterocins
present an activity spectrum narrower than Type 2 and
broader than Type 3 enterocins produced by E. faecalis.
Enterocins, as most bacteriocins, have the cytoplasmic
membrane as their primary target. They form pores in the cell
membrane, thereby depleting the transmembrane potential and/
or the pH gradient, resulting in the leakage of indispensable
intracellular molecules (Cleveland et al., 2001).
2.2. Citrate metabolism by enterococci
Citrate is present in many raw materials that are used in food
fermentations, such as fruit, vegetables, and milk, and it is also
used as an additive for the production of fermented sausages.
The behavior of LAB may differ from one species to another,
and not all LAB are able to metabolize citrate. The ability to
metabolize citrate is invariably linked to endogenous plasmids
that contain the gene encoding the transporter, which is
responsible for citrate uptake from the medium. Since citrate
is a highly oxidized substrate, no reducing equivalents, such as
NADH, are produced during its degradation, which results in
the formation of metabolic end products other than lactic acid.
These include diacetyl, acetaldehyde, acetoin, and 2,3-butanediol,
which have very distinct aroma properties and significantly
influence the quality of fermented foods (Hugenholtz,
1993). For instance, diacetyl determines the aromatic properties
of fresh cheese, fermented milk, cream, and butter. The
breakdown of citrate also results in the production of carbon
dioxide, which can contribute to the texture of some fermented
dairy products.
Most of the knowledge on the metabolic pathways involved
in citrate metabolism has been derived from LAB of dairy
origin, although citrate itself does not always support their
growth. This claim is based on the inability of certain LAB to
grow in batch cultures to which excess citrate was added as the
sole energy source. Nevertheless, most LAB are able to cometabolize
citrate in the presence of another energy source,
such as glucose or lactose. The metabolism of citric acid has
been extensively studied and is well documented in strains of
Lactococcus, Lactobacillus, and Leuconostoc (Drinan et al.,
1976; Cogan, 1987; Kennes et al., 1991; Starrenburg and

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M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1 – 24
Table 1
Enterocins produced by enterococci classified as class I, class II, or class III bacteriocins
Class I Producer strain Amino acid sequence of the purified enterocin Reference a
Cyl L L E. faecalis DS16 MENLSVVPSFEELSVEEMEAIQGSGDVQAETTPVQ
Gilmore et al., 1994
CAVAATAAASSAACGWVGGGIFTGVTVVVSLKHC
Cyl L S E. faecalis DS16 VLNKENQENYYSNKLELVGPSFEELSLEEMEAIQGQ
SGDVQAETTPACFTIGLGVGALFSAKFC
Gilmore et al., 1994
Class IIa Producer strain MM b Amino acid sequence of the purified enterocin pI c Reference
Enterocin A E. faecium CTC492 4833 TTHSGKYYGNGVYCTKNKCTVDWAKATTQ
9.0 Aymerich et al., 1996
CIAGMSIGGFLGGAIPGKC
Enterocin CRL 35 E. faecium CRL 35 KYYGNGVTLNKXGXSVNXXXA... Farías et al., 1996
Bacteriocin 31 E. faecalis YI717 5009 ATYYGNGLYCNKQKCWVDWNKASREIGKIIQ
9.7 Tomita et al., 1996
VNGWVQHGPWAPR
Enterocin P E. faecium P13 4630 ATRSYGNGVYCNNSKCWVNWGEAKENIAGIQ
8.3 Cintas et al., 1997
VISGWASGLAGMGH
Mundticin E. mundtii AT06 4290 KYYGNGVSCNKKGCSVDWGKAIGIIGNNSAQ
9.8 Bennik et al., 1998
ANLATGGAAGWSK
Bacteriocin RC714 E. faecium RC714 4937 ATYYGNGLYCNKEKCWVDWNQAKGEIGKIIQ
9.3 del Campo et al., 2001
VNGWVNHGPWAPR
Enterocin SE-K4 E. faecalis K-4 4918 ATYYGNGVYCNKQKCWVDWSRARSEIIDRGQ
VKAYVNGFTKVLG
9.6 Eguchi et al., 2001
Class IIc Producer strain MM Amino acid sequence of the purified enterocin pI Reference
Enterocin AS-48 E. faecalis S-48 7167 MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVQ
GSGGLSLLAAAGRESIKAYLKKEIKKKGKRAVIAW
10.6 Martínez-Bueno
et al., 1994
Enterocin B E. faecium T136 5465 ENDHRMPNELNRPNNLSKGGAKCGAAIAGGLFGIPQ 9.6 Casaus et al., 1997
KGPLAWAAGLANVYSKCN
Enterocin L50A E. faecium L50 5190 MGAIAKLVAKFGWPIVKKYYKQIMQFIGEGWAINQ 10.4 Cintas et al., 1998b
KIIEWIKKHI
Enterocin L50B E. faecium L50 5178 MGAIAKLVTKFGWPLIKKFYKQIMQFIGQGWTIDQ 10.7 Cintas et al., 1998b
QIEKWLKRH
Enterocin EJ97 E. faecalis EJ97 5328 MLAKIKAMIKKFPNPYTLAAKLTTYEINWYKQQQ
YGRYPWERPVA
10.8 Gálvez et al., 1998;
Sánchez-Hidalgo et al., 2003
Enterocin Q E. faecium L50 3952 MNFLKNGIAKWMTGAELQAYKKKYGCLPWEKISC 9.7 Cintas et al., 2000
Enterocin 1071A E. faecalis BEF 1071 4286 ESVFSKIGNAVGPAAYWILKGLGNMSDVNQAQ
DRINRKKH
10.3 Balla et al., 2000;
Franz et al., 2002
Enterocin 1071B E. faecalis BEF 1071 3899 GPGKWLPWLQPAYDFVTGLAKGIGKEGNKNKWKNV 10.4 Balla et al., 2000;
Franz et al., 2002
Enterocin RJ-11 E. faecalis RJ-11 5049 APAGLVAKFGRPIVKKYYKQIMQFIGEGSAINKIIPQ
WIARMWRT
11.1 Yamamoto et al., 2003
Class III Producer strain MM Amino acid sequence of the purified enterocin pI Reference
Enterolysin A E. faecalis LMG 2333 34501 ASNEWSWPLGKPYAGRYEEGQQFGNTAFNRGGTYQ
FHDGFDFGSAIYGNGSVYAVHDGKILYAGWDPVGGQ
GSLGAFIVLQAGNTNVIYQEFSRNVGDIKVSTGQTVQ
KKGQLIGKFTSSHLHLGMTKKEWRSAHSSWNKDDQ
GTWFNPIPILQGGSTPTPPNPGPKNFTTNVRYGLRVQ
LGGSWLPEVTNFNNTNDGFAGYPNRQHDMLYIKVDQ
KGQMKYRVHTAQSGWLPWVSKGDKSDTVNGAAGMQ
PGQAIDGVQLNYITPKGEKLSQAYYRSQTTKRSGWLQ
KVSADNGSIPGLDSYAGIFGEPLDRLQIGISQSNPF
9.5 Nilsen et al., 2003
a Reference dealing with the actual purification and amino acid sequence analysis.
b MM (Da) calculated theoretically from the amino acid sequence (http://www.basic.nwu.edu/biotools/proteincalc.html).
c pI calculated theoretically from the amino acid sequence (http://www.embl-heidelberg.de/cgi/pi-wrapper.pl).
Hugenholtz, 1991; Hugenholtz et al., 1993; Palles et al., 1998;
Kimoto et al., 1999; Goupry et al., 2000). In contrast, more
limited and sporadic data concerning citrate metabolism by
Enterococcus strains are available (Coventry et al., 1978;
Hagrass et al., 1991; Urdaneta et al., 1995; Freitas et al., 1999).
Nevertheless, during the last few years more studies have
focused on citrate metabolism by enterococci (Sarantinopoulos
et al., 2001a,b, 2003; Rea and Cogan, 2003a,b). This increased
interest in citrate metabolism by Enterococcus strains has
probably arisen due to the fact that enterococci, in particular the
major species E. faecium and E. faecalis, frequently occur in
large numbers in many cheeses, especially those manufactured
in the Mediterranean area. Milk citrate catabolism by enterococci
may explain, among other mechanisms, the role of
enterococci in the development of the distinctive organoleptic
properties of these cheeses.

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 5
Table 2
Enterocins described in the literature similar to existing enterocins
Enterocin Actual enterocin Producer strain Reference
Enterocin 4 Enterocin AS-48 E. faecalis INIA 4 Joosten et al., 1996
Enterococcin EFS2 E. faecalis EFS2 Maisnier-Patin et al., 1996
Bacteriocin 21 E. faecalis OG1X Tomita et al., 1997
Enterocin 7C5 E. faecium 7C5 Folli et al., 2003
Enterocin 900 Enterocin A E. faecium BFE 900 Franz et al., 1999b
Enterocin 1146 E. faecium DPC1146 O’Keeffe et al., 1999
Enterocin EFM01 E. faecium EFM01 Ennahar and Deschamps, 2000
Enterocin P21 E. faecium P21 Herranz et al., 2001
Enterocin 81 E. faecium WHE 81 Ennahar et al., 2001
Enterocin N15 E. faecium N15 Losteinkit et al., 2001
Enterocin BC25 E. faecium BC25 Morovský et al., 2001
Enterocin CCM 4231 E. faecium CCM 4231 Foulquié Moreno et al., 2003a
Enterocin 900 Enterocin B E. faecium BFE 900 Franz et al., 1999b
Enterocin P21 E. faecium P21 Herranz et al., 2001
Enterocin 81 E. faecium WHE 81 Ennahar et al., 2001
Enterocin RZS C13 E. faecium RZS C13 Foulquié Moreno et al., 2003a
Enterocin RZS C5 E. faecium RZS C5 Foulquié Moreno et al., 2003a
Enterocin I Enterocin L50 E. faecium 6T1a Floriano et al., 1998
Enterocin B2 E. faecium B2 Moreno et al., 2002
Enterocin AA13 Enterocin P E. faecium AA13 Herranz et al., 1999
Enterocin G16 E. faecium G16 Herranz et al., 1999
Enterocin B1 E. faecium B1 Moreno et al., 2002
Enterocin B2 E. faecium B2 Moreno et al., 2002
Enterocin 1071A Enterocin 1071A E. faecalis FAIR-E 309 Franz et al., 2002
Enterocin 1071B Enterocin 1071B E. faecalis FAIR-E 309 Franz et al., 2002
Enterocin B2 Enterocin 1071 E. faecium B2 Moreno et al., 2002
Almost 60 years ago, Campbell and Gunsalus (1944) and
Gunsalus and Campbell (1944) showed that pH had a very
significant effect on product formation from citrate in
enterococci. Twenty-five years later, Devoyod (1969) pinpointed
that E. faecalis subsp. liquefaciens had the ability to
metabolize citrate in the absence of carbohydrates. Since then
there appears to have been little work on citrate metabolism by
enterococci, until Hagrass et al. (1991) studied two strains of E.
faecalis, isolated from a fermented milk product. They showed
that compounds, such as acetaldehyde and diacetyl, were
produced via citrate metabolism. Raffe (1994) observed that E.
faecalis strains, grown in skim milk, could produce lactic acid
from lactose, and acetic acid from citrate. During the same
period, Urdaneta et al. (1995) studied citrate metabolism in
three E. faecalis strains, grown in synthetic media, having
citrate as the sole energy source. All strains not only grew in
those media, but they also produced acetate as the final
product. Moreover, Freitas et al. (1999) concluded that E.
faecalis and E. faecium strains, which were grown in sheeps’
and goats’ milk, produced relatively high amounts of lactic
acid and lower amounts of formate and acetate, indicating
indirectly that lactose and citrate were co-metabolised.
Recently, Sarantinopoulos et al. (2001b) showed that E.
faecalis FAIR-E 229, a strain isolated from Cheddar cheese,
was able to grow in skim milk, and co-metabolism of lactose
and citrate took place. Moreover, when increasing initial
concentrations of citrate were used as the sole energy source,
the main end products were acetate and formate, as a result of
citrate consumption. Surprisingly, the same strain did not
utilize citrate in MRS broth containing lactose instead of
glucose, and in MRS broth containing glucose with increasing
citrate concentrations. This phenomenon was explained by
Rea and Cogan (2003a) who studied the factors affecting cometabolism
of citrate and glucose by growing and resting
cells of three E. faecalis and two E. faecium strains. It was
suggested that the presence of glucose prevents citrate
utilization through catabolite repression, so that citrate can
only be utilized when all of the glucose in the medium is
consumed. The same authors suggested that catabolite
repression by glucose and fructose occurs in E. faecalis
strains, but this is not the case when galactose or sucrose are
used as energy sources (Rea and Cogan, 2003b). In contrast,
Sarantinopoulos et al. (2003) observed co-metabolism of
glucose and citrate for an E. faecium strain, grown in MRS
broth, both under aerobic and anaerobic conditions and
uncontrolled pH. These results fully support the findings of
Sarantinopoulos et al. (2001a) who, after an extensive
screening of 129 E. faecalis, E. faecium and E. durans
strains for general biochemical properties, showed that the
above set of strains exhibited strain-to-strain variation
concerning citrate metabolism, but with the majority of the
E. faecalis strains able to utilize citrate.
2.3. Proteolysis and lipolysis by enterococci
2.3.1. Proteolysis
The degradation of casein plays a major role in the
development of texture in cheese. In addition, some peptides
contribute to the formation of flavour, whereas other,
undesirable bitter-tasting peptides can lead to off-flavours.

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Also, the secondary degradation of amino acids is considered
to have a major impact on the flavour development in cheese.
Besides the indigenous milk proteolytic enzymes and rennin
used for protein coagulation, LAB cell wall-associated
proteinases and intracellular peptidases released after cell
lysis in the curd are considered to play an important role in
casein hydrolysis during cheese preparation (Wilkinson et al.,
1994).
Studies performed so far concerning proteolysis by LAB are
mainly restricted to the genera Lactococcus and Lactobacillus
(Pritchard and Coolbear, 1993; Sousa et al., 2001). The
biochemical pathways, which lead to casein degradation and
peptide/amino acid transportation, are believed to be the same
in the case of the other LAB, including the genus Enterococcus.
However, only sporadic data exist in the literature
regarding the proteolytic system of enterococci, in comparison
with other LAB species. Even though there are no commonly
accepted laboratory methods in the literature for determining
proteolytic and peptidolytic activities, such activities are
generally low for Enterococcus strains, with the exception of
E. faecalis strains (Dovat et al., 1970; Arizcun et al., 1997;
Macedo and Malcata, 1997; Suzzi et al., 2000; Andrighetto et
al., 2001).
The very first work concerning the proteolytic system of
enterococci was performed by Somkuti and Babel (1969). They
studied an extracellular proteinase, which was produced by an
E. faecalis var. liquefaciens strain. This proteinase was able to
hydrolytically degrade casein in an intensive way and it was
less active against h-lactoglobulin and a-lactalbumin. In
another relevant study during that period, Wallace and Harmon
(1970) examined an intracellular protease of an E. durans
strain. The protease was particularly active against casein and
h-lactoglobulin, but it was not able to hydrolyze bovine serum
albumin. Furthermore, Dovat et al. (1970) studied the
proteolytic activity of enterococci in milk and showed that
they exhibited low proteolytic activity, with the exception of
strains belonging to the species E. faecalis var. liquefaciens.
Almost 20 years later, Carrasco de Mendoza et al. (1989)
determined in a more systematic study the extracellular
proteolytic activity of 61 Enterococcus strains using sodium
caseinate as substrate. They suggested that the proteolytic
activity was strain- and time-dependent. After 48 h of
incubation, strains belonging to E. faecalis species were more
proteolytic, and this was more evident after 120 h. Furthermore,
Wessels et al. (1990) examined 108 E. faecium, E.
faecalis, and E. durans strains, isolated from various dairy
products. A large percentage of these strains of all species were
able to grow at 7 -C, and more than half of them showed
proteolytic activity at psychrotrophic temperatures.
In another work, Hegazi (1990a) studied the proteolysis of S.
faecalis subsp. liquefaciens 3/6 in skim milk with some
additives. It was suggested that calcium lactate and some
inorganic phosphate salts do not influence casein hydrolysis. In
contrast, calcium carbonate, which is frequently used as an
additive in cheese manufacturing, resulted in a markedly
reduction of hydrolysis levels. Also, low NaCl concentrations
(2%, w/v) positively influenced casein breakdown, while higher
concentrations inhibited it. The same author concluded that the
activity of an extracellular proteinase produced by the same
strain (S. faecalis subsp. liquefaciens 3/6) was reduced when
the strain was grown in milk that was processed at high
temperature (Hegazi, 1990b). This specific proteinase was able
to hydrolyze casein, h-lactoglobulin and a-lactalbumin. The
activity of the enzyme was strongly reduced in the presence of
EDTA and for that reason it was considered a metalloenzyme.
At the same time, Garcia de Fernando et al. (1991a,b)
examined in depth the extracellular proteinase production by E.
faecalis subsp. liquefaciens. The microorganism grew well and
developed extracellular proteinase activity on proteinaceous
media. The molar mass of the enzyme has been estimated to be
approximately 30 kDa by Sephadex gel filtration and approximately
26 kDa by sodium-dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE). The isoelectric point has
been found to be 4.6 and maximum enzyme activity of the
proteinase has been observed at pH 7.5 and 45 -C. The enzyme
hydrolyzed casein, bovine serum albumin, a-lactalbumin and
h-lactoglobulin. Macedo et al. (2000) suggested that an E.
faecium strain isolated from Serra cheese exhibited neglecting
aminopeptidolytic and endopeptidolytic activity in synthetic
media, in comparison with strains belonging to the species of
L. paracasei, L. lactis, and Leuconostoc mesenteroides.
Furthermore, Villani and Coppola (1994) examined the
proteolytic activity of 24 E. faecium and 60 E. faecalis strains,
after growth in skim milk, at 37 -C for 6 h. All E. faecalis
strains were much more proteolytic, in comparison with the E.
faecium strains. Quite recently, Andrighetto et al. (2001)
showed that the majority of the 124 enterococcal strains
studied, isolated from traditional Italian cheeses, displayed
weak proteolytic activities in milk, but 30 of them belonging to
the E. faecalis species were more proteolytic. Finally, the same
conclusion was drawn in another systematic study performed
by Sarantinopoulos et al. (2001a), who screened 129 E.
faecium, E. faecalis, and E. durans strains for biochemical
properties relevant to their technological performance. It was
found that all strains exhibited low extracellular proteolytic and
peptidolytic activities, with the E. faecalis strains being
generally more active.
2.3.2. Lipolysis
Lipids have a major effect on the flavour and texture of
cheese. They contribute to cheese flavour in three ways: (i)
being a source of fatty acids, especially short-chain fatty acids,
which may be converted to other sapid and aromatic
compounds, especially methyl ketones and lactones; (ii) being
a cause of oxidation of fatty acids, leading to the formation of
various unsaturated aldehydes that are strongly flavored and
thus cause a flavour defect referred to as oxidative rancidity;
(iii) being solvents for sapid and aromatic compounds,
produced not only from lipids, but also from proteins and
lactose. Milk fat hydrolysis during cheese manufacture and
ripening is due to the endogenous milk lipase, the lipolytic
enzymes of starter cultures and non-starter bacteria, lipases
from psychrotrophic bacteria, and–depending on the cheese
variety–exogenous enzyme preparations (El Soda et al., 1995).

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 7
LAB are generally considered to be weakly lipolytic, as
compared with other groups of microorganisms. However, it is
believed that lipolytic activity by LAB plays an important role
in the determination of the special aroma of many different
cheeses. Certain LAB, which are used as bulk starter cultures
(Lactococcus and Lactobacillus), together with some other
bacteria (Leuconostoc, Enterococcus, Pediococcus, and Micrococcus),
which can survive pasteurization and/or contaminate
cheese during maturation, are believed to significantly
contribute to cheese fat hydrolysis (El Soda et al., 1995;
Collins et al., 2003). Concerning the lipolytic activity of
enterococci, limited and often contradictory data exist in the
literature.
The first work regarding the lipolytic and esterolytic
activities of enterococci was performed by Lund (1965), who
determined electrophoretically the presence of esterases in cellfree
extracts of E. faecalis, E. faecium and E. durans strains.
The electrophoretic pattern of the E. faecalis strains was
different compared to the patterns of the other two species.
Moreover, E. faecalis strains exhibited higher activity, as
determined on the basis of the intensity of the esterolytic bands.
At the same time, Dovat et al. (1970) studied the lipolytic
activity of 16 Enterococcus and 5 Streptococcus strains, after
they had grown in skim milk, milk cream and skim milk in the
presence of tributyrin, using as substrate Nile Blue Sulphate
Agar. It was concluded that enterococci were more lipolytic.
The hydrolysis rate reduced with increasing molecular mass
(tripropionin>tributyrin>tricaprin >tricaprylin), while triolein
was not hydrolyzed at all. In the same study, the volatile fatty
acids produced were chromatographically determined, showing
that enterococci, and especially the E. durans strains, produced
more acetic acid than the Streptococcus strains.
A few years later, Martínez-Moreno (1976) showed that
Enterococcus strains (E. durans, E. faecium, and E. faecalis)
isolated from Manchego cheese, were active only against
tributyrin and not against milk fat. Chander et al. (1979a,b) in
two consecutive studies examined the influence of certain fatty
acids on the growth of and lipase production by E. faecalis, and
they purified and characterized this lipase. It was observed that
low-molecular-mass fatty acids (C3, C4, C6, and C10)
enhanced lipase production, in contrast to high-molecular-mass
fatty acids (C12, C14, C16, C18, and C18:2) that inhibited
lipase production. The molar mass of this specific enzyme has
been estimated to be approximately 21 kDa and the isoelectric
point has been found to be 4.6. The maximum enzyme activity
of the lipase has been observed at pH 7.5 and 40 -C. The
enzyme was able to hydrolyze tributyrin more than tricaproin,
tricaprylin, and triolein.
After 10 years of silence on the lipolytic system of
enterococci, new studies have been published. Carrasco de
Mendoza et al. (1992) concluded that the lipolytic activity of
enterococci in milk was strain-dependent. Most of the strains
examined exhibited low activity and only a few strains
belonging to E. faecalis species were characterized as lipolytic.
In the same period, Tsakalidou et al. (1993) used synthetic
substrates to detect photometrically and post-electrophoretically
the esterolytic activities of E. faecium and E. durans. E.
durans strains were active against low-molecular-mass fatty
acids up to 4-nitrophenyl-caprylate (C8), while E. faecium was
active up to 4-nitrophenyl-stearate (C18). Differences between
the two species were also obvious for the post-electrophoretic
detection of esterases. Moreover, Tsakalidou et al. (1994a)
described the purification and characterization of an intracellular
esterase of an E. faecium strain isolated from Feta cheese.
The enzyme with a molecular mass of 45 kDa was optimally
active at 35 -C and pH 8.0. It could hydrolyze synthetic
substrates of low and medium molecular mass (C2 to C12)
with the highest activity against 4-nitrophenyl-butyrate (C4).
Tsakalidou et al. (1994b) observed that enterococci generally
exhibited higher esterolytic activities than other LAB
species. In contrast, Villani and Coppola (1994) reported that
enterococci showed low lipolytic activity when grown in whole
milk. However, Tavaria and Malcata (1998) suggested that an
E. faecium strain isolated from Serra da Estrela cheese was able
to hydrolyze milk fat more extensively than a Lactococcus
lactis subsp. lactis strain. In addition, Hemati et al. (1998)
reported that enterococci isolated from Domiati cheese
exhibited greater esterolytic activity in comparison with
Lactobacillus strains.
In another much more systematic study, Sarantinopoulos et
al. (2001a) showed that, among 129 enterococci, the majority
(90%) hydrolyzed all substrates from tributyrin (C4) to
tristearin (C18) with a decreasing extent. In addition,
concerning esterases, all strains were active on the synthetic
substrates used, from 4-nitrophenyl-acetate (C2) to -stearate
(C18), with decreasing activity. Generally, E. faecalis strains
were the most lipolytic and esterolytic, followed by the E.
faecium and E. durans strains. Finally, while enterococci
isolated from Beyaz cheese showed a pronounced lipolytic
activity (Durlu-Ozkaya et al., 2001), the ones isolated from
Semicotto Caprino cheese were not able to hydrolyse
tributyrin, independent of the species (Suzzi et al., 2000).
2.4. Enterococci as probiotics
The definition of a probiotic has been evolving with the
increasing understanding of the mechanisms by which they
influence human health (De Vuyst et al., 2004). The term was
first introduced to describe substances produced by one
microorganism (protozoa) that stimulate the growth of other
microorganisms (Lilly and Stillwell, 1965). Nowadays, the
definition most commonly used is that of Fuller (1989), i.e.
‘‘probiotics are live microbial feed supplements, which
beneficially affect the host animal by improving its intestinal
microbial balance’’. In 1992, the definition was expanded to
include food and non-food uses as well as the use of mono and
mixed cultures (Havenaar and Huis in’t Veld, 1992). Hence, a
probiotic can be described as a preparation of or a product
containing viable, defined microorganisms in sufficient numbers,
which alter the microbiota (by implantation or colonization)
in a compartment of the host and that exert beneficial
health effects in this host (De Vuyst et al., 2004). They are
applied in foods such as yogurt, fermented and non-fermented
milks, infant formulas, and pharmaceutical preparations.

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Probiotic consumption is reported to exert a myriad of
beneficial effects, including enhanced immune response,
balancing of colonic microbiota, vaccine adjuvant effects,
reduction of fecal enzymes implicated in cancer initiation,
treatment of diarrhea associated with travel and antibiotic
therapy, control of rotavirus and Clostridium difficile-induced
colitis, and prevention of ulcers related to Helicobacter pylori.
Probiotics are also implicated in the reduction of serum
cholesterol, the antagonism against food-borne pathogens and
tooth decay organisms, the amelioration of lactose malabsorption
symptoms as well as candidiasis and urinary tract
infections (Saavedra, 2001). A group of requirements have
been identified for a microorganism to be defined as an
effective probiotic (Salminen et al., 1996). These include the
ability to (a) adhere to cells; (b) exclude or reduce pathogenic
adherence; (c) persist and multiply; (d) produce acids,
hydrogen peroxide, and bacteriocins antagonistic to pathogen
growth; (e) be safe, noninvasive, noncarcinogenic, and
nonpathogenic; and (f) coaggregate to form a normal balanced
flora.
Strains that are used as probiotics for man have been
isolated from the human gastrointestinal tract and usually
belong to species of the genera Lactobacillus and Bifidobacterium.
However, strains belonging to species of other LAB,
among them enterococci, have been used in the past as
probiotics too, such as E. faecium, E. faecalis, S. thermophilus,
L. lactis subsp. lactis, Leuconostoc mesenteroides, Propionibacterium
freudenreichii, Pediococcus acidilactici, Sporolactobacillus
inulinus, E. coli, Bacillus cereus (Ftoyoi_), and the
yeast Saccharomyces cerevisiae (Fboulardii_) (Fuller, 1989;
O’Sullivan et al., 1992; Holzapfel et al., 1998). Even though
strains of E. faecium and E. faecalis species have been applied
in human, probiotic supplements, E. faecalis strains have also
been widely used as veterinary feed supplements. Since
February 2004, 10 preparations (9 different strains of E.
faecium) are authorized as additives in feeding stuffs in the
European Union (European Commission, 2004).
A well-studied Enterococcus strain used as probiotic is E.
faecium SF 68, which is produced in Switzerland (Cylactin\
LBC SF68 ME10, F. Hoffmann-La Roche Ltd., Basel,
Switzerland). It has been proposed to be clinically effective
in the prevention of antibiotic-associated diarrhea (Wunderlich
et al., 1989) and in the treatment of diarrhea in children
(Bellomo et al., 1980). E. faecium SF 68 has been tested in
adults with acute diarrhea in two hospitals in Belgium
(Buydens and Debeuckelaere, 1996). Although this type of
diarrhea was self-limited (>95% recovered by 6 days), the use
of the enterococci shortened the duration of diarrhea by 1–3
days. E. faecium SF 68 has also been studied as a feed
probiotic. E. faecium SF 68 used in a dry dog food
significantly enhanced cell-mediated and humoral immune
functions in dogs (Benyacoub et al., 2003). In Denmark, a
fermented milk product that contains E. faecium SF 68 (GAIO;
MD Foods, Aarhus, Denmark) has been sold for several years
because of its hypocholesterolemic effect on individuals
(Agerbaek et al., 1995). However, two long-term studies (12
weeks and 6 months, respectively) failed to show any reduction
in cholesterol levels in individuals who received GAIO,
compared with that in control subjects at the end of the
treatment (Richelsen et al., 1996; Sessions et al., 1997). In a
recent work, the same fermented milk product GAIO was used
to study the relationship of the E. faecium SF 68-containing
probiotic and a vancomycin treatment. It was concluded that E.
faecium SF 68 from the GAIO product was not found to
acquire vancomycin resistance during the study period (Lund et
al., 2000).
Rossi et al. (1999) used the strain E. faecium CRL 183 for
the development of a novel fermented soymilk product with
potential probiotic properties. E. faecium CRL 183 in
combination with Lactobacillus jugurti resulted in a 43%
decrease in cholesterol in vitro. Also, the probiotic strain E.
faecium PR88 (E. faecium Fargo 688\ from Quest International,
Naarden, The Netherlands) was studied in a human
clinical trial. The consumption of this strain led to alleviation of
the symptoms of irritable bowl syndrome in humans (Allen et
al., 1996). The strain was used for the production of a probiotic
Cheddar cheese. It did not affect cheese composition, but an
improvement of the flavour of the cheese was obtained
(Gardiner et al., 1999).
Another probiotic product available on the market that
contains an E. faecium strain is Walthers ECOFLOR (Walthers
Health Care, Den Haag, The Netherlands). The producer claims
the efficacy of the E. faecium strain against diarrhea, its anticarcinogenic
effect, the production of enterocins active towards
L. monocytogenes, the possible decrease of the LDL-cholesterol
level, as well as its sensitivity to vancomycin and the
production of L(+) lactic acid (personal communication).
However, the producer provides no scientific evidence.
The use of enterococci as probiotics remains a controversial
issue. While the probiotic benefits of some strains are well
established, the emergence and the increased association of
enterococci with human disease and multiple antibiotic
resistances (see below) have raised concern regarding their
use as probiotics. The fear that antimicrobial resistance genes
or genes encoding virulence factors can be transferred to other
bacteria in the gastrointestinal tract contributes to this
controversy (Franz et al., 2003).
3. Pathogenesis of enterococci
Enterococci are among the most common nosocomial
pathogens and they have been implicated as an important
cause of endocarditis, bacteremia, and infections of the urinary
tract, central nervous system, intra-abdominal and pelvic
infections, as well as of multiple antibiotic resistances (Endtz
et al., 1999; Franz et al., 1999a). Several virulence factors have
been described (Jett et al., 1994) and the number of antibioticresistant
enterococci (ARE), especially vancomycin-resistant
enterococci (VRE), is increasing (Endtz et al., 1999).
3.1. Cytolytic activity of enterococci
In 1949, Sherwood et al. reported on some h-hemolytic
group D streptococci that inhibited growth of other bacteria. In

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 9
the following years, other research groups observed the same
phenomenon (for reviews, see Jett et al., 1994; Haas and
Gilmore, 1999). The evidence that bacteriocin and hemolytic
activities were provided by a single compound came when both
activities were simultaneously lost after exposure to UV
irradiation but were simultaneously regained by reversion
(Brock and Davies, 1963). Granato and Jackson (1969,
1971a,b) found convincing data that E. faecalis hemolysin/
bacteriocin was a natural bifunctional component. For simplicity,
the hemolysin/bacteriocin produced by E. faecalis has been
termed cytolysin (Gilmore et al., 1990). Amino acid analysis of
the purified E. faecalis cytolysin revealed that it belongs to the
class I lantibiotics (Gilmore et al., 1994). Cytolysin is unique
among the lantibiotics in that it is able to lyse eukaryotic cells in
addition to its bactericidal effect. As a class I lantibiotic,
cytolysin forms pores in the cytoplasmic membrane of bacterial
target cells. Sequence and complementation analysis revealed a
gene cluster containing six genes that are required for cytolysin
production (Gilmore et al., 1994; Saris et al., 1996). The two
genes that encode the cytolysin subunits are designated cylL L
(encoding for a peptide of 68 amino acids) and cylL S (encoding
for a peptide of 63 amino acids). Only the strains that are able to
express and secrete both subunits are hemolytic (Booth et al.,
1996). The cytolysin genes are located on the highly transmissible
pheromone-responsive conjugative plasmid pAD1 (Ike et
al., 1990). Pheromones are small peptides (seven to eight amino
acids) secreted by E. faecalis that promote conjugative transfer
of plasmids between strains (Ike and Clewell, 1984). These
peptides are chromosomally encoded and are referred to as
pheromones because they elicit a specific mating response from
plasmid-carrying donor cells. Typically, multiple pheromones
are secreted simultaneously by a given E. faecalis strain. In
addition to pheromones, each pheromone-responsive plasmid
encodes a secreted peptide that acts as a competitive inhibitor of
its corresponding pheromone (Jett et al., 1994).
Cytolysin is considered a virulence factor of E. faecalis
strains in animal models (Ike et al., 1984; Huycke et al., 1991;
Jett et al., 1992; Chow et al., 1993; Singh et al., 1998).
However, the role of this factor in enterococcal pathogenicity
remains unclear. In a recent study, E. faecalis isolated from
patients with endocarditis or bacteremia and from healthy
volunteers were investigated for their ability to adhere to Int-
407 and Girarti heart cell lines (Archimbaud et al., 2002). No
link between the presence of some virulence factors such as
gelatinase, aggregation substances, and cytolysin, and the
ability of the strains to adhere to these cells could be found.
3.2. Antibiotic resistance of enterococci
Antibiotic resistance encompasses both natural (intrinsic)
resistance and acquired (transferable) resistance. Enterococci
possess a broad spectrum of antibiotic resistances within these
two types (Klare et al., 2001). Examples of intrinsic resistance
are vancomycin (VanC type) resistance in E. gallinarum, and
resistance towards streptogramins in E. faecalis, as well as
resistance to isoxazolylpenicillins, cephalosporins, monobactams,
aminoglycosides (low level), lincosamides (mostly), and
polymyxins. The resistance to ampicillin (especially in E.
faecium), tetracyclines, macrolides, aminoglycosides (high
level), chloramphenicol, trimethoprim/sulfamethozaxole, quinolones,
and streptogramins (in E. faecium and related species)
are acquired, as well as resistance towards glycopeptides
(vancomycin). VRE possess vanA, vanB, vanD, and vanE type
resistance genes (Klare et al., 2001). Ampicillin, vancomycin
and gentamicin are the most clinically relevant antibiotics to
cure infections with multiple ARE strains. It should be noted
that the extensive use of vancomycin has steadily raised the
number of VRE over the past two decades and therefore the
percentage of invasive nosocomial enterococci displaying
high-level vancomycin resistance (Endtz et al., 1999). However,
antibiotic resistance as such cannot explain the virulence
of enterococci. Unfortunately, VRE are also highly resistant to
all standard anti-enterococcal drugs (Landman and Quale,
1997), and, therefore, VRE constitute a serious risk group.
Dutka-Malen et al. (1995) developed a PCR assay to detect
glycopeptide resistance genotypes.
Glycopeptide-resistant enterococci are phenotypically and
genotypically heterogeneous. Of the six different VRE phenotypes
known to date (Leclercq et al., 1988, 1992; Quintiliani et
al., 1993; Perichon et al., 1997; Fines et al., 1999; McKessar et
al., 2000), phenotypes VanA and VanB are of the highest
clinical importance as they are most frequently observed in two
predominant enterococcal species, E. faecalis and E. faecium
(Murray, 1997, 1998; Cetnikaya et al., 2000). VanA-type strains
display high-level inducible resistance to both vancomycin and
teicoplanin, following the acquisition of transposon Tn1546 or
closely related mobile genetic elements (Arthur et al., 1993).
VanB-type strains display variable levels of inducible resistance
to vancomycin only (Arthur et al., 1996).
ARE are widespread in food. They have been found in meat
products, dairy products, and ready-to-eat foods, and even
within enterococcal strains used as probiotics (Franz et al.,
2001; Giraffa, 2002). Franz et al. (2001) found acquired
resistance traits for a number of antibiotics in enterococci from
food origin throughout Europe. However, in general these
strains did not show resistance to the clinically relevant
antibiotics. Giraffa (2002) emphasizes the role played by
ARE, and especially VRE, as possible natural food reservoirs
in the dissemination of antibiotic-resistant traits in the environment.
There could be a link between the use of some antibiotics
in livestock and humans becoming colonised by ARE via the
food chain. For safety reasons, a critical or important criterion to
check is the absence of transferable antibiotic resistance in
enterococci that could be used as co-cultures or starter cultures
in foods, since it is strain-specific (Franz et al., 2001;
Vancanneyt et al., 2002; De Vuyst et al., 2003).
3.3. Virulence factors of enterococci
A virulence factor is an effector molecule that enhances the
ability of a microorganism to cause disease beyond that
intrinsic to the species background (Mundy et al., 2000).
Typical examples are aggregation substance, gelatinase, extracellular
superoxide, and extracellular surface protein.

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3.3.1. Aggregation substance
Aggregation substance (Agg) is a pheromone-inducible
surface protein of E. faecalis, which promotes aggregate
formation during bacterial conjugation (Clewell, 1993). As an
important component of the bacterial pheromone-responsive
genetic exchange system, Agg mediates efficient enterococcal
donor–recipient contact to facilitate plasmid transfer (Clewell,
1993). This trait may contribute to the pathogenesis of
enterococcal infection through different mechanisms. The
cells that express this trait form large aggregates in vivo.
However, it is unknown how this influences phagocytosis and
subsequent damage of the vital functions of the organism.
Also, Agg may bind on and present its cognate ligand to the
surface of the organism, possibly resulting in superantigen
activity. Finally, Agg increases the hydrophobicity of the
enterococcal surface, which may induce localization of
cholesterol to phagosomes, and prevent or delay fusion with
lysosomal vesicles (Mundy et al., 2000). Studies on endocarditis
showed synergism between cytolysin and Agg. Up to
now, Agg is exclusively found in E. faecalis strains; however,
its incidence among food isolates seems to be high (Eaton
and Gasson, 2001; Franz et al., 2001).
3.3.2. Gelatinase
Gelatinase (Gel) is an extracellular metallo-endopeptidase
involved in the hydrolysis of gelatin, collagen, haemoglobin,
and other bioactive peptides (Su et al., 1991). The association
between an enterococcal protease and virulence was first
suggested in 1975 by Gold et al., who found that a gelatinliquefying,
human, oral E. faecalis isolate induced caries
formation in germ-free rats, while non-proteolytic strains did
not. Su et al. (1991) sequenced the protease gene (gelE) inE.
faecalis. Singh et al. (1998) demonstrated that Gel, which is
commonly produced by nosocomial, fecal, and clinical
enterococcal isolates, is a virulence factor of enterococci, at
least for peritonitis in mice. The presence of Gel production
among food E. faecalis strains is high (Eaton and Gasson,
2001; Franz et al., 2001). However, Eaton and Gasson (2001)
demonstrated that even when the gel gene is present, a
negative phenotype can be found. None of the E. faecium
strains involved in both studies produce Gel.
3.3.3. Extracellular surface protein
Extracellular surface protein (Esp) was first described in a
clinical E. faecalis MMH594 isolate by Shankar et al. (1999).
The esp gene is unusually large consisting of 5622 nucleotides
capable of encoding a primary translation product of
1873 amino acids, with a theoretical molecular mass of
approximately 202 kDa. Studies on the distribution of this
surface protein revealed a significant enrichment in infectionderived
E. faecalis isolates (Shankar et al., 1999; Waar et al.,
2002). Esp is thought to play a role in adhesion and evasion
of the immune response of the host. The incidence of Esp in
food isolates differs for E. faecium and E. faecalis. While it
is hardly found in E. faecium, the incidence in E. faecalis is
higher than expected. No explanation could be found up to
now (Eaton and Gasson, 2001; Franz et al., 2001).
3.4. Opsonophagocytic assay to assess the safety of potential
enterococcal pathogens
Opsonophagocytic killing is used as an in vitro test to detect a
protective immune response against bacterial pathogens (Pier et
al., 1994), as all the antibodies that provide antibacterial
immunity are opsonic. E. faecalis and E. faecium can posses a
capsular polysaccharide that is the target for opsonic antibodies
(Huebner et al., 1999). Hufnagel et al. (2003) compared one
probiotic E. faecalis strain with a collection of clinical isolates
and found that 89% of the clinical strains were less susceptible to
killing mediated by normal rabbit sera. The results showed a
strain-dependent susceptibility to opsonic killing. Moreover,
opsonophagocytic killing is considered to be an important test to
assess the safety of enterococcal strains (Hufnagel et al., 2003).
4. Enterococci in food
Enterococci are widely distributed in the environment,
principally inhabiting the human and animal gastrointestinal
tract. E. faecalis is often the predominating Enterococcus
species in the human bowel, although in some individuals and
in some countries E. faecium outnumbers E. faecalis (Devriese
and Pot, 1995). However, Mundt (1986) demonstrated that the
common presence of E. faecalis in many food products is not
always related to direct faecal contamination. In 1992, the EU
established a maximum level for the presence of coliforms and
Escherichia coli, both considered as indicators of hygiene,
while no limit was set for the enterococci (Anonymous, 1992).
Furthermore, it has been shown that enterococci had little value
as hygiene indicators in the industrial processing of foods
(Birollo et al., 2001).
Moreover, although E. faecalis, E. faecium, and E. durans
are frequently isolated from human faeces, they are much less
prevalent in livestock, such as pigs, cattle, and sheep (Franz
et al., 1999a). Enterococci are not only associated with warmblooded
animals, but they also occur in soil, surface waters,
and on plants, vegetables, and insects (Deibel and Silliker,
1963; Mundt, 1986). The resistance of enterococci to
pasteurization temperatures, and their adaptability to different
substrates and growth conditions (low and high temperature,
extreme pH, and salinity) implies that they can be found
either in food products manufactured from raw materials
(milk or meat) and in heat-treated food products. This means
that these bacteria could withstand usual conditions of food
production. In addition, they can contaminate finished
products during food processing. Therefore, enterococci can
become an important part of the fermented food microbiota,
especially in fermented cheeses and meats.
The contribution of enterococci to the organoleptic properties
of fermented food products and their ability to produce
bacteriocins (enterocins) are important characteristics for their
application in food technology. In recent years, the reports
about enterococci used as starter cultures or co-cultures
(adjuncts) have increased considerably. The major part of this
kind of research has focused on the applicability of Enterococcus
strains, which harbor interesting biochemical and

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 11
technological properties, in cheese products and, to less extent,
in meat products. However, the selection of enterococci to be
involved in food processes is a difficult task, due to their
potential risk in human health (Vancanneyt et al., 2002; De
Vuyst et al., 2003).
4.1. Cheese products
4.1.1. Presence of enterococci in cheese
Enterococci are associated with traditional European
cheeses manufactured in Mediterranean countries, such as
Greece, Italy, Spain and Portugal, from raw or pasteurized
goats’, ewes’, water-buffalos’ or bovine milk (Ordoñez et al.,
1978; Trovatelli and Schliesser, 1987; Coppola et al., 1988; Del
Pozo et al., 1988; Litopolou-Tzanetaki, 1990; Litopoulou-
Tzanetaki and Tzanetakis, 1992; Poullet et al., 1993; Kalogridou-Vassiliadou
et al., 1994; Torri Tarelli et al., 1994;
Macedo et al., 1995; Centeno et al., 1996; Cuesta et al., 1996;
Zárate et al., 1997; Aran, 1998; Bouton et al., 1998; Pérez
Elortondo et al., 1999; Xanthopoulos et al., 2000; Sarantinopoulos
et al., 2002a; Manolopoulou et al., 2003). The
prevalence of enterococci in milk and cheese has long been
considered a result of unhygienic conditions during the
production and processing of milk, such as direct contamination
by animal and human feaces. Their presence has been
indirectly linked with contaminated water sources, the exterior
of animals, the milking equipment, and bulk storage tanks
(Gelsomino et al., 2001, 2002; Giraffa, 2002).
Levels of enterococci in different cheese curds range from
10 4 to 10 6 CFU g 1 and in the fully ripened cheeses from 10 5 to
10 7 CFU g 1 (Del Pozo et al., 1988; Litopolou-Tzanetaki,
1990; Litopoulou-Tzanetaki and Tzanetakis, 1992; Poullet et
al., 1993; Kalogridou-Vassiliadou et al., 1994; Macedo et al.,
1995; Centeno et al., 1996; Cuesta et al., 1996; Zárate et al.,
1997; Franz et al., 1999a; Pérez Elortondo et al., 1999;
Xanthopoulos et al., 2000; Sarantinopoulos et al., 2001a; Franz
et al., 2003; Giraffa, 2002; Manolopoulou et al., 2003), with E.
faecium and E. faecalis being the dominant species. More
interestingly, in cheeses such as Manchego (Ordoñez et al.,
1978), Mozzarella (Coppola et al., 1988), Kefalotyri (Litopolou-Tzanetaki,
1990), Picante de Beira Baixa (Freitas et al.,
1995), Serra (Macedo et al., 1995), Cebreiro (Centeno et al.,
1996), Comté (Bouton et al., 1998), Domiati (Hemati et al.,
1998), and Kashar (Aran, 1998), enterococci are the predominant
microorganisms in the fully ripened product, while
according to the results of a 15-year survey, enterococci ranged
from 10 4 to 10 6 CFU g 1 in Emmental cheese and from 10 4 to
10 7 CFU g 1 in Appenzeller cheese (Teuber et al., 1996). The
European cheeses in which enterococci have been studied are
compiled in Table 3.
Varying levels in different cheeses result from the cheese
type, the production season, the extent of milk contamination,
Table 3
European cheeses where enterococci have been studied
Origin Cheese Type of milk Reference
French Comté Cow Bouton et al., 1998
French Roquefort Cow Devoyod, 1969
French Venaco Ewe/goat Casalta and Zennaro, 1997
Greek farmhouse cheese Ewe Gomez et al., 2000
Greek Feta Ewe Litopolou-Tzanetaki et al., 1993
Greek Feta and Teleme Ewe Tzanetakis and Litopoulou-Tzanetaki, 1992
Greek Kefalotyri Ewe Litopolou-Tzanetaki, 1990
Greek Orinotyri Ewe Prodromou et al., 2001
Greek Pichtogalo Chanion Ewe/goat Papageorgiou et al., 1998
Irish Cheddar Cow Gelsomino et al., 2001
Italian Buffalo Mozzarella Buffalo Parente et al., 1989
Italian Fiore Sardo Sheep Ledda et al., 1994
Italian Fontina Cow Battistotti et al., 1977
Italian Montasio Cow Basso et al., 1994
Italian Monte Veronese Cow Torriani et al., 1998
Italian Mozzarella Cow Morea et al., 1999
Italian Pecorino Sardo Ewe Mannu et al., 1999; Mannu and Paba, 2002
Italian Semicotto Caprino Goat Suzzi et al., 2000
Portuguese Serra da Estrela Ewe Macedo et al., 1995
Portuguese Serra da Estrela Ewe Tavaria and Malcata, 1998
Spanish Armada Goat Tornadijo et al., 1995
Spanish Arzúar Cow Centeno et al., 1995
Spanish Cebreiro Cow Centeno et al., 1996, 1999
Spanish Cebreiro Cow Menéndez et al., 1998
Spanish Majorero Goat Fontecha et al., 1990
Spanish La Serena Ewe Fernandez del Pozo et al., 1988
Spanish Manchego Ewe Ramos et al., 1981
Spanish Roncal-Idiazabal Ewe Arizcun et al., 1997
Spanish San Simón Cow García et al., 2002
Spanish Tenerife goat cheese Goat Zárate et al., 1997
Spanish Tetilla Cow Menéndez et al., 2001

12
M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1 – 24
and survival in the dairy environment (dependent on seasonal
temperature), as well as survival and growth under the
particular conditions of cheese manufacture and ripening
(Litopolou-Tzanetaki, 1990; Litopoulou-Tzanetaki and Tzanetakis,
1992; Macedo et al., 1995; Sarantinopoulos et al.,
2001a).
Although some authors have claimed in the past that high
levels of contaminating enterococci could lead to deterioration
of sensory properties in some cheeses (Thompson and Marth,
1986; López-Diaz et al., 1995), other literature reports discuss
the beneficial role of the presence of enterococci in cheese.
Enterococci contribute to the ripening and aroma development
of these products due to their proteolytic and esterolytic
activities, as well as the production of diacetyl and other
important volatile compounds (Jensen et al., 1975a,b; Ordoñez
et al., 1978; Trovatelli and Schliesser, 1987; Centeno et al.,
1996, 1999; Casalta and Zennaro, 1997; Franz et al., 1999a,
2003; Sarantinopoulos et al., 2001a,b). Furthermore, enterococci
seem to play an important role in improving flavor
development and quality, not only of cheese, but also of other
traditional fermented foods, such as vegetables and sausages
(Franz et al., 1999a; Giraffa, 2002).
4.1.2. Applicability of enterococci as starter or adjunct cultures
in cheese
The presence of enterococci in high numbers in many
different types of cheeses, their potential contribution to the
organoleptic properties of fermented food products, and their
ability to produce bacteriocins (enterocins) are important
characteristics to be considered by food processors for their
direct application in food manufacture. During the last few
years, the reports about enterococci proposed to be used as
starter cultures or co-cultures (adjuncts) have considerably
increased. The aim of these studies was to elucidate the role that
enterococci play in cheese ripening. However, as enterococci
have been recognized in recent years as major nosocomial
pathogens, one should carefully consider the potential virulence
factors of this group of microorganisms before use.
It has been proposed that enterococci can be included as part
of defined starter cultures for different European cheeses. The
effect of enterococci as starter cultures or co-cultures was
studied in cheeses, such as Cheddar (Dahlberg and Kosikowsky,
1948; Czulak and Hammond, 1956; Jensen et al.,
1975a,b), Feta (Litopolou-Tzanetaki et al., 1993; Sarantinopoulos
et al., 2002a), water-buffalo Mozzarella (Coppola et al.,
1988; Parente et al., 1989; Villani and Coppola, 1994), Venaco
(Casalta and Zennaro, 1997), Cebreiro (Centeno et al., 1996,
1999), and Hispánico (Garde et al., 1997; Oumer et al., 2001).
In most of these studies, E. faecalis strains have been used, and
only in the case of Feta cheese, strains belonging to the species
of E. faecium (Sarantinopoulos et al., 2002a) and E. durans
(Litopolou-Tzanetaki et al., 1993) have been tested. Also, the
British FAdvisory Committee on Novel Foods and Processes_
(ACNFP) accepted the use of E. faecium strain K77D as a
starter culture in fermented dairy products (ACNFP, 1996).
In an early, comparative study, Jensen et al. (1975b) used
two E. faecalis strains and two E. durans strains as adjunct
starters for the production of Cheddar cheese. They concluded
that the cheese batches manufactured with the addition of E.
faecalis strains exhibited greater proteolytic degradation in
comparison to the cheese batches manufactured without
enterococci or with the addition of E. durans strains. More
recently, when E. durans strains were used as co-cultures in the
production of Feta cheese and a white-brined cheese, the
proteolytic index was higher in the experimental cheese than in
the control. That was justified by the fact that the degree and
the formation rate of non-casein nitrogen (NCN, pH 4.6 soluble
nitrogen) and of phosphotungistic acid (PTA)-soluble nitrogencontinuously
increased with ripening time (Litopolou-Tzanetaki
et al., 1993; Tzanetakis et al., 1995). An increased watersoluble
nitrogen content and proteolytic index was also
observed when E. faecalis strains were used as adjunct starters
in cheeses such as Cebreiro (Centeno et al., 1999), Hispánico
(Garde et al., 1997; Oumer et al., 2001) and Venaco (Casalta
and Zennaro, 1997).
More recently, Centeno et al. (1999) examined the effects of
E. faecalis in Cebreiro cheese manufacture. It has been
concluded that h-casein was broken down to a greater extent
in the batches containing E. faecalis strains than in the control
ones. Moreover, the highest level of a s1 -casein hydrolysis and
the highest ratio of peptide a s1 -I/a s1 -casein was obtained when
E. faecalis strains were used. In all batches made with
enterococci, volatile free fatty acids, long-chain free fatty
acids, and diacetyl and acetoin were higher than in the control
batches. Increase of free fatty acids in cheese containing
enterococci was seen in Cheddar (Jensen et al., 1975a) and Feta
cheese (Sarantinopoulos et al., 2002a) too. However, Centeno
et al. (1999) recommended the use of moderate proteolytic (and
lipolytic) strains of E. faecalis to guarantee the quality of
traditional Cebreiro cheese.
Finally, Sarantinopoulos et al. (2002a) studied in detail the
effect of two E. faecium strains, as adjunct starter cultures, on
the microbial, physicochemical and sensory characteristics of
Feta cheese. Both enterococcal strains positively affected taste,
aroma, color, and structure of the full-ripened cheeses, as well
as the overall sensory profile. These results supported those
obtained by Litopolou-Tzanetaki et al. (1993) when E. durans
strains were used for the production of Feta cheese.
4.1.3. Applicability of bacteriocinogenic enterococci and/or
their enterocins in cheese
There are many studies concerning the isolation and
characterization of bacteriocins (enterocins) produced by
Enterococcus spp. Enterocins have been isolated from strains
originating from different sources, including foods (cheese,
meat, fish, and vegetables), animals, and humans. Table 4
shows a compilation of bacteriocinogenic Enterococcus strains
(Bac + ) from different origins that have been studied. The
enterococcal bacteriocins are usually active towards foodborne
pathogens such as Listeria spp. and Clostridium spp. (Ben
Embarek et al., 1994; Farías et al., 1994; Vlaemynck et al.,
1994; Giraffa et al., 1994, 1995a,b; Maisnier-Patin et al., 1996;
Vlaemynck, 1996; Cintas et al., 1998a; Bennik et al., 1999;
Mendoza et al., 1999). Activity towards Gram-negative

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 13
Table 4
Origin of bacteriocinogenic Enterococcus strains
Source Strain(s) Reference
Dairy products E. faecium DPC1146 Parente and Hill, 1992a
E. faecium 7C5 Torri Tarelli et al., 1994
E. faecium RZS C5 Vlaemynck et al., 1994
E. faecalis INIA 4 Joosten et al., 1995
E. faecium CRL 35 Farías et al., 1996
E. faecium WHE 81 Ennahar et al., 1998
Screening for Bac + strains Carnio et al., 1999
Screening for enterocin Rodríguez et al., 2000
AS-48 producer strains
E. faecalis TAB 28 Rodríguez et al., 2000
Screening for Bac + strains Elotmani et al., 2002
E. faecalis FAIR-E 309 Franz et al., 2002
E. faecium FAIR E-198 Sarantinopoulos et al., 2002b
Screening for Bac + strains Saavedra et al., 2003
Fermented
sausages
Other fermented
foods
E. faecium L1 Lyon et al., 1995
E. faecium CTC492 Aymerich et al., 1996
E. faecalis EFS2 Maisnier-Patin et al., 1996
E. faecium T136 Casaus et al., 1997
E. faecium P13 Cintas et al., 1997
E. faecium L50 Cintas et al., 1995, 1998b
E. faecium AA13 Herranz et al., 1999
E. faecium G16 Herranz et al., 1999
E. faecium P21 Herranz et al., 2001
E. casseliflavus IM 416K1 Sabia et al., 2002
E. faecium N15 Losteinkit et al., 2001
E. faecium B1 Moreno et al., 2002
E. faecium B2 Moreno et al., 2002
Screening for Bac + strains Onda et al., 2002
Screening for Bac + strains Yamamoto et al., 2003
Fish Screening for Bac + strains Ben Embarek et al., 1994
Vegetables E. faecalis 226 Villani et al., 1993
E. faecium BFE 900 Franz et al., 1996
E. mundtii ATO6 Bennik et al., 1998
E. faecium 6T1a Floriano et al., 1998
Silage Screening for Bac + Kato et al., 1994
E. faecium strains
E. faecium RZS C13 Vlaemynck et al., 1994
E. faecium NIAI 157 Ohmomo et al., 2000
E. faecalis K-4 Eguchi et al., 2001
Veterinary E. faecium CCM 4231 Lauková et al., 1993
E. faecium BC25 Morovský et al., 1998
E. faecium J96 Audisio et al., 1999
E. faecalis BFE 1071 Balla et al., 2000
Screening for Bac + strains du Toit et al., 2000
E. gallinarum 012 Jennes et al., 2000
Water E. faecalis EJ97 Gálvez et al., 1998
Human origin E. faecalis S-48 Gálvez et al., 1986
E. faecium YI717 Tomita et al., 1996
Screening for Bac + strains del Campo et al., 2001
E. faecium RC714 del Campo et al., 2001
bacteria such as E. coli (Gálvez et al., 1989; Tomita et al.,
1997) and Vibrio cholerae (Simonetta et al., 1997) has been
shown as well. Furthermore, Wachsman et al. (1999) reported
on the antiviral activity of enterocin CRL35.
For the above-mentioned reasons the use of bacteriocinproducing
enterococci as starter cultures, co-cultures (adjunct
cultures) or protective cultures, as well as the direct addition of
the enterocins in food fermentation processes, is a point of
great interest amongst fermentation technologists. As a
consequence, the applicability of such enterococci cultures
(Table 5) and/or the corresponding enterocins produced (Table
6) has been studied in a wide variety of fermented food
products (mainly in cheese products) at small, pilot-scale
conditions. However, the functionality of such strains at largescale,
industrial, food processing conditions is still unknown
and certainly needs to be thoroughly examined in the near
future.
Reports on the optimization of enterocin production and
growth of enterococci during batch fermentation are scarce.
Nevertheless, kinetic studies of batch fermentations can be
used to analyze the relation between bacteriocin production,
growth of the producer organism, and the microbial environment.
Such studies have indicated that the bacteriocin
production by E. faecium RZS C5 follows a growthassociated
process and is limited to the early growth phase
because of a shut off mechanism (Leroy and De Vuyst, 2002;
Leroy et al., 2003a). Moreover, they indicated that the
kinetics of E. faecium RZS C5 are affected by the pH and
the temperature, and the concentration of sugar, complex
nutrients, and sodium chloride (Leroy and De Vuyst, 2002;
Leroy et al., 2003b). Another very well studied bacteriocin is
enterocin 1146 from E. faecium DPC 1146 (Parente and Hill,
1992a,b,c; Parente and Ricciardi, 1994a,b; Parente et al.,
1997; Foulquié Moreno et al., 2003b). Enterocin 1146 has a
rapid bactericidal effect on L. monocytogenes in buffer
systems, broth and milk, and it is stable during the ripening
period of Cheddar cheese, as it is the case for E. faecium
FAIR-E 171 (Foulquié Moreno et al., 2003b). As enterocin
production by E. faecium FAIR-E 171 takes place from the
beginning of the cheese production and is stable during the
whole cheese-ripening phase, both early and late contamination
of the milk or cheese by Listeria spp. can be combated
with stable, in situ enterocin production.
Some successful examples of in situ enterocin production
during cheese making have been described and the stability
of certain enterocins during cheese ripening has been
reported in Taleggio, Manchego, and Cheddar cheese
(Giraffa et al., 1994, 1995b; Giraffa and Carminati, 1997;
Núñez et al., 1997; Foulquié Moreno et al., 2003b). Giraffa
et al. (1994) showed that enterococci are suitable to produce
bacteriocins in milk, in the presence of rennet, during an
incubation that simulated the first 55 h of Taleggio cheese
making. Núñez et al. (1997) concluded that E. faecalis INIA
4 is able to produce its enterocin in competition with the
milk native microbiota during the manufacture of Manchego
cheese. Furthermore, an increase in enterocin activity was
recorded in the cheese during the first week. During
Taleggio cheese manufacture and ripening, Giraffa et al.
(1995b) showed that bacteriocin production by E. faecium
7C5 starts during whey drainage and generally reaches a
maximum amount just after cheese salting. The stability of
the enterocin during ripening is not affected, although the
heterogeneous microbiota present in raw milk, as well as the
enzymatic activity of rennet, are capable to inactivate the
inhibitor. Furthermore, E. faecium 7C5 inhibits the growth of
L. monocytogenes Ohio on the surface of Taleggio cheese
(Giraffa and Carminati, 1997).

14
M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1 – 24
Table 5
Applications of enterocin producing strains
Strain Enterocin produced Application Reference
E. faecalis B114 Enterocin not known Camembert cheese Sulzer and Busse, 1991
E. faecium 7C5 Enterocin 7C5 Taleggio cheese Giraffa et al., 1995b
E. faecalis INIA 4 Enterocin 4 Manchego cheese Joosten et al., 1995
E. faecalis INIA 4 Enterocin 4 Hispano cheese Garde et al., 1997
E. faecalis INIA 4 Enterocin 4 Manchego cheese Núñez et al., 1997
E. faecalis INIA 4 Enterocin 4 Hispano cheese Oumer et al., 2001
E. faecium CCM 4231 Enterocin CCM 4231 Saint-Paulin cheese Lauková et al., 2001
E. faecium CCM 4231 Enterocin CCM 4231 Spanish-style dry fermented sausages Callewaert et al., 2000
E. faecium RZS C13 Enterocin RZS C13 Spanish-style dry fermented sausages Callewaert et al., 2000
E. faecium CTC492 Enterocins A and B Dry fermented sausages Aymerich et al., 2000b
E. faecium CTC492 Enterocins A and B Cooked pork Aymerich et al., 2002
E. faecalis TAB 28 Enterocin AS-48 Raw milk cheese Rodríguez et al., 2001
E. faecium RZS C5 Enterocin RZS C5 Cheddar cheese production Foulquié Moreno et al., 2003b
E. faecium DPC 1146 Enterocin DPC 1146 Cheddar cheese production Foulquié Moreno et al., 2003b
E. faecium FAIR-E 198 Enterocins A and/or P Feta cheese Sarantinopoulos et al., 2002b
E. casseliflavus IM 416K1 Enterocin 416K1 Italian sausage (Cacciatore) Sabia et al., 2003
Although E. faecium FAIR-E 198 was able to grow well
under the conditions resembling Feta cheese manufacture,
enterocin production was low or negligible. Moreover and
in contrast with the enterocin 1146, no enterocin activity
was detected when the same strain was used as an adjunct
starter culture for Feta cheese production (Sarantinopoulos
et al., 2002b). The latter phenomenon can be attributed to
the fact that enterocin production in milk using enterococci
as co-cultures can be lower than when enterococci are used
as pure cultures (Giraffa et al., 1995a,b; Sarantinopoulos et
al., 2002b; Foulquié Moreno et al., 2003b). Another
explanation could be that the enterocin is not produced at
all under the specific conditions of this technology or that it
is produced but can not be detected due to adsorption on
the cheese matrix. This phenomenon has been reported
before (Sulzer and Busse, 1991). Farías et al. (1999)
studied the effect of enterocin CRL35 used as additive
during goat cheese making contaminated with Listeria.
Although enterocin CRL35 could not be detected during
ripening, suppression of Listeria took place during the
whole period of ripening.
Table 6
Applications of enterocins as additives
Enterocin Application Reference
Enterocin 226 NWC Mozzarella cheese Villani et al., 1993
Enterocin 4 A model dairy system Rodríguez et al., 1997
Enterocin CCM 4231 Cattle slurry environment Lauková et al., 1998
Enterocin CCM 4231 Soy milk Lauková and Czikkova,
1999
Enterocin CCM 4231 Dry fermented Hornád
salami
Lauková et al., 1999
Enterocin CCM 4231
Bryndza, a traditional
Slovak dairy product
from sheep milk
Lauková and Czikková,
2001
Enterocin CRL 35 Goat cheese making Farías et al., 1999
Enterocin CRL 35 Meat system Vignolo et al., 2000
Enterocin CTC 492 Meat products Aymerich et al., 2000b
Enterocin CTC 492 Cooked pork Aymerich et al., 2002
4.2. Meat products
4.2.1. Presence of enterococci in meat
The presence of enterococci in the gastrointestinal tract of
animals may lead to contamination of meat at the time of
slaughtering. In a study of enterococci from raw meat products,
E. faecalis was the predominant isolate from beef and pork cuts
(Stiles et al., 1978). E. faecium was also frequently isolated
from Bologna, a processed pack sausage (Stiles et al., 1978).
Pig carcasses from three different slaughtering plants contained
mean log counts of 10 4 –10 8 enterococci per 100 cm 2 of
carcass surface. E. faecium and E. faecalis were the predominant
species isolated (Knudtson and Hartman, 1993). E.
faecalis predominated the Gram-positive cocci isolated from
chicken samples collected at poultry abattoirs. Enterococci
were consistently isolated from beef, poultry, and pig carcasses,
or from fresh meat in studies of antibiotic ARE (Franz et al.,
2003).
Besides raw meats, enterococci are also associated with
processed meats. Heating of processed meats during production
may confer a selective advantage to enterococci because these
bacteria are among the most thermotolerant of the nonsporulating
bacteria. After surviving the heat-processing step,
both E. faecalis and E. faecium have been implicated in
spoilage of cured meat products, such as canned hams and
chub-packed luncheon meats (Magnus et al., 1986, 1988). This
is especially true where recontamination with competing
bacteria is prevented, i.e. when products are heated after
packaging in cans or in impermeable plastic films. The heat
resistance of enterococci in these products is influenced by
components, such as salt, nitrite, and meat tissue (Bell and
DeLacey, 1984; Magnus et al., 1986, 1988; Franz et al.,
1999a). To prevent spoilage of the processed meats by
enterococci, it was suggested that initial contamination by
these microorganisms should be kept to a minimum, and that
adequate heat processing should be based on D-values of the
most heat-resistant enterococci isolated from raw materials
(Magnus et al., 1986).

M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 15
Enterococci have also been isolated from some types of
fermented sausages. Salami and Landjäger were shown to
contain enterococci at numbers ranging from 100 to 2.6 10 5
CFU g 1 (Teuber et al., 1996). Enterococci were isolated from
dry fermented sausages such as Chorizo (Casaus et al., 1997;
Cintas et al., 1997) and Espetec (Aymerich et al., 1996), both
produced in Spain. Moreover, in German and Italian fermented
sausages, Marchesino et al. (1992) reported concentrations of
enterococci from 10 3 to 10 5 CFU g 1 at the end of the ripening
process in sausages manufactured both with and without starter
cultures.
In Mediterranean countries, many traditional and artisan
fermented meat products are manufactured mostly by small
companies, farms, or local butchers. Usually, they are low-acid
products with a pH higher than standard sausages (pH>5.3). In
these types of products, the fermentation is carried out without
the use of starter cultures, relying on the endogenous flora to
keep the traditional organoleptic qualities. As a consequence,
the natural microbiota is constituted by a mixture of species of
LAB including enterococci and lactobacilli, as well as
coagulase-negative staphylococci and yeasts. These products
have a long history of safe consumption by humans. In a
survey of 31 naturally fermented sausages, purchased at several
supermarkets in the northeast of Spain, the number of
enterococci ranged from 1.3 to 4.48 log CFU g 1 , the
population of total LAB being from 7.12 to 9.07 log CFU
g 1 (Hugas et al., 2003).
The biochemical activities of enterococci in a sausage
matrix have not been studied as in the case of dairy products.
They might contribute to sausage aromatisation by their
glycolytic, proteolytic, and lipolytic activities. Furthermore,
metmyoglobin reduction activity has been described for meat
enterococci (Hugas et al., 2003).
4.2.2. Applicability of bacteriocinogenic enterococci and/or
their enterocins in meat products
Even though there are many bacteriocin-producing enterococcal
strains isolated from various fermented, meat products
(Table 4), only a very limited number of studies has been
conducted aimed at the direct application of such strains in the
production of meat products (Tables 5 and 6). Very recently,
some attempts have been made using E. faecium strains as
adjunct starter cultures or their enterocins for the manufacture
of fermented, meat products.
E. faecium RZS C13 and E. faecium CCM 4231 were
used as starter cultures for the production of Spanish-style
dry, fermented sausages (Callewaert et al., 2000). The
Enterococcus strains were partially competitive during meat
fermentation and strongly inhibited the growth of Listeria
spp. As no production of off-flavours was detected, the
enterococci might be suitable for addition to meat as cocultures
to improve food safety. Aymerich et al. (2000b)
applied the enterocins A and B produced by E. faecium CTC
492 as semi-purified food additives in different meat
products. An inhibitory effect was observed on the growth
of Listeria. However, the addition of the producer strain as
the sole starter culture in dry fermented sausages had no
effect on the inhibition of Listeria growth (Aymerich et al.,
2000a). In contrast, the combination of both E. faecium CTC
492 and its enterocins can prevent ropiness in sliced cooked
ham (Aymerich et al., 2002).
Furthermore, the antilisterial efficiency of enterocins
CRL35 (produced by E. faecium CRL35) has been tested in
broth and a meat system against L. monocytogenes and L.
innocua (Vignolo et al., 2000). Both Listeria species initially
decreased in viable counts followed by regrowth of the
survivors after 1 h in the presence of the enterocins. The
organoleptic properties of the meat model system were not
affected. Finally, the effectiveness of enterocin CCM 4231 in
controlling L. monocytogenes contamination of dry fermented
Hornád salami has been examined (Lauková et al., 1999). The
enterocin addition resulted in the reduction of L. monocytogenes
by 1.67 logs when compared to the control immediately
after the addition of bacteriocin. Although on the second day
the growth of the pathogen in the experimental sample reached
3.38 log CFU g 1 , a difference of 1.72 log was found, in
comparison with the control. After one week of ripening the L.
monocytogenes count in the control reached 10 7 CFU g 1 ,
while in the experimental sample the count was 10 4 CFU g 1 ,
a difference that was maintained after 2 and 3 weeks of
ripening.
As stated before and as found also in many other application
studies, it has been shown that there are limitations to the
usefulness of some enterocins as antilisterial agents, as full
suppression of the pathogen is rarely achieved in fermented
food systems (e.g. cheeses). The optimal use of bacteriocins for
inhibiting the growth of Listeria and other foodborne pathogens
or food spoilage microorganisms requires detailed
knowledge of their activity against sensitive strains and the
factors that may limit their effectiveness. These factors can be
revealed through a thorough examination of the foods’
structure and composition and their interaction with bacteriocins.
As a consequence, there is a great need to maximize the
amount of the bioavailable enterocin during the food fermentation
process, by selecting enterococcal strains that are
characterized by a naturally high bacteriocin production
capacity, or, much more importantly, which are well adapted
to the food environment in which they are to be used
(Sarantinopoulos et al., 2002b).
4.3. Vegetables and olives
Enterococci commonly occur in large numbers in vegetables,
olives, and plant material (Mundt, 1963; Fernández-Diaz,
1983; Franz et al., 1999a; Giraffa, 2002; Ben Omar et al.,
2004). However, literature data concerning the isolation and
characterization of enterococci from such products is scarce.
Enterococci have been isolated from Spanish-style green olive
fermentations (Fernández-Diaz, 1983; Floriano et al., 1998; de
Castro et al., 2002), in which E. faecalis and E. faecium are
frequent contaminants. In a very recent study, Ben Omar et al.
(2004) studied the functional properties, the virulence characteristics,
and the antibiotic resistance of fifty enterococcal
isolates from different Spanish food samples. Three of them

16
M.R. Foulquié Moreno et al. / International Journal of Food Microbiology 106 (2006) 1 – 24
were isolated from olives and belonged to the species E.
faecium. Moreover, it was found that the viable counts of
enterococci from olives obtained on Slanetz and Bartley
medium was 2.210 3 CFU g 1 .
In another very recent study, Randazzo et al. (2004) isolated
a total number of 52 LAB from naturally fermented green
olives collected from different areas of Sicily. Even though the
majority of these strains belonged to the genus of Lactobacillus,
four of them were identified as Enterococcus spp. on the
basis of phenotypic and biochemical traits. Characterization
using restriction fragment length polymorphism (RFLP)
analysis of 16S rDNA revealed that these four strains belonged
to the species E. faecium, E. casseliflavus, and E. hirae. To our
knowledge there is only one study dealing with the role of
enterococci during the fermentation of table olives (de Castro et
al., 2002). In this study, strain Enterococcus casseliflavus cc45,
isolated from the fermentation brine, in combination with strain
Lactobacillus pentosus CECT 5138 of the same origin, were
used as starter cultures in Spanish-style green olive fermentation.
The best results were attained when cultures were used
sequentially, enterococci at day 1 and lactobacilli at day 2. This
type of inoculation produced a quicker brine acidification, a
greater consumption of carbohydrates, and a rapid pH decrease.
The diversity of vegetable products available on the
European market is increasing, and minimally processed,
ready-to-eat type products are popular among consumers. As
these products may pose a health risk, bacteriocinogenic LAB or
purified bacteriocins may be employed to improve their safety.
Nevertheless, the use of bacteriocins, produced by enterococcal
strains, as biopreservatives in vegetables and olives has been
studied to a very small extent (Villani et al., 1993; Franz et al.,
1996; Bennik et al., 1998; Floriano et al., 1998).
5. Conclusions
Enterococci are widespread in nature and are present in dairy
and other fermented food products. They directly contribute to
the typical taste and flavor of traditional Mediterranean cheeses,
as they can grow in a cheese environment. They also seem to
play a role in other traditional fermented foods such as sausages,
olives, and vegetables. Their capacity to produce bacteriocins
active towards spoilage or pathogenic bacteria, in particular
Listeria, might be a powerful tool for the protection of these
products, for instance in the case of raw milk cheeses, for the
production of traditional cheeses with an improved aroma, or
for the production of medium-pH fermented sausages. Also,
enterococci have been used as probiotics because of their
possible health-promoting capacities. However, some enterococci
carry potential virulence factors and can display pathogenic
traits. Furthermore, the number of VRE has been
increasing during the last decade. Fortunately, these important
features are strain-dependent. For these reasons, the selection of
Enterococcus strains of interest in the food industry should be
based on the absence of any possible pathogenic properties, or
transferable antibiotic resistance genes. Modern analysis techniques
as well as up-to-date knowledge on these strains and
their properties will help the food processor and the consumer to
accept enterococci as other LAB as an important player in
certain food products.
Acknowledgments
This work was carried out in the framework of the FAIR-
CT97-3078 project ‘‘Enterococci in Food Fermentations:
Functional and Safety Aspects’’. MRFM was recipient of a
Marie Curie Fellowship (grant FAIR-CT97-5013). PS was
supported by the State Scholarships Foundation of Greece
(IKY-IDrima Kratikon Ypotrofion). LDV acknowledges the
financial support from the Research Council of the VUB and
the Fund for Scientific Research-Flanders.
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