International Journal of Food Microbiology 106 (2006) 1 – 24 www.elsevier.com/locate/ijfoodmicro Review The role and application of enterococci in food and health

M.R. Foulquie´ 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

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: [email protected] (L. De Vuyst). 1 M.R. Foulquie´ 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 2 M.R. Foulquie´ 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 columbae grow poorly or not at all in the presence of 6.5% NaCl (Devriese et al., 1990, 1993; Vancanneyt et al., 2004). The genus Enterococcus belongs to a group of micro- Most enterococcal species can hydrolyse aesculin in the organisms known as lactic acid bacteria (LAB). Enterococci presence of 40% bile salts. Enterococcal species possess the are Gram-positive, non-sporeforming, catalase-negative, ox- Lancefield group D antigen with the exception of E. idase-negative, facultative anaerobic cocci that occur singly, cecorum, E. columbae, E. dispar, E. pseudoavium, E. in pairs, or in chains. From a taxonomic point of view, the saccharolyticus and E. sulfureus (Hardie and Whiley, genus Enterococcus has been reviewed several times 1997). E. mundtii and E. casseliflavus are yellow-pigmented, (Schleifer and Kilpper-Ba¨lz, 1987; Stackebrandt and Teuber, and E. gallinarum and E. casseliflavus are motile (Schleifer 1988; Devriese and Pot, 1995; Hardie and Whiley, 1997). In and Kilpper-Ba¨lz, 1987). 1984, DNA–DNA hybridisation and 16S rRNA sequencing studies indicated that the species Streptococcus faecium and 2. Functional properties of enterococci S. faecalis were sufficiently distinct from other streptococci, and Schleifer and Kilpper-Ba¨lz (1984) proposed their Studies on the microbiota of traditional cheeses in the transfer to the genus Enterococcus. To date, 28 species, Mediterranean countries, which are mainly produced from namely E. asini, E. avium, E. canis, E. casseliflavus, E. raw ewes’ or goats’ milk, and less commonly from cows’ cecorum, E. columbae, E. dispar, E. durans, E. faecalis, E. milk, have indicated that enterococci play an important role faecium, E. flavescens, E. gallinarum, E. gilvus, E. in the ripening of these cheeses, probably through proteol- haemoperoxidus, E. hirae, E. malodoratus, E. moraviensis, ysis, lipolysis, and citrate breakdown, hence contributing to E. mundtii, E. pallens, E. phoeniculicola, E. pseudoavium, their typical taste and flavour, (Coppola et al., 1990; E. raffinosus, E. ratti, E. saccharolyticus, E. saccharomi- Litopoulou-Tzanetaki and Tzanetakis, 1992; Ledda et al., nimus, E. solitarius, E. sulfureus, and E. villorum, have 1994; Giraffa et al., 1997; Manolopoulou et al., 2003). They been added to the genus on the basis of phylogenetic are also present in other fermented food products, such as evidence strengthened by 16S rRNA DNA sequencing and/ sausages (Franz et al., 1999a, 2003; Giraffa, 2002; Hugas et or DNA–DNA hybridization studies. However, some reclas- al., 2003) and olives (Ferna´ndez-Diaz, 1983; Franz et al., sifications may occur in the near future. For instance, E. 1996, 1999a; Floriano et al., 1998; Ben Omar et al., 2004). flavescens and E. casseliflavus have insufficient differences Enterococci have the ability to produce bacteriocins, the so- to be classified separately (Devriese and Pot, 1995; called enterocins, which are small peptides with antimicro- Descheemaeker et al., 1997), and E. avillorum is an earlier bial activity towards closely related Gram-positive bacteria heterotypic synonym of E. porcinus (De Graef et al., 2003). including spoilage or pathogenic bacteria, such as Listeria Enterococci grow optimally at a temperature of 35 -C, (De Vuyst and Vandamme, 1994). Moreover, enterococci are although most species in the genus will grow at temperatures used in some countries as probiotics (Franz et al., 1999a, ranging from 10 to 45 -C. They can also grow in the 2003). 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. 2.1. Bacteriocin production by enterococci sulfureus (Martı´nez-Murcia and Collins, 1991), E. malodor- atus (Collins et al., 1984) and E. moraviensis (Svec et al., The bacteriocins produced by LAB belong to the large 2001), which do not grow at 45 -C, and E. cecorum and E. group of ribosomally synthesised, small, cationic, amphiphilic columbae, which do not grow at 10 -C(Devriese et al., (rather hydrophobic), antimicrobial peptides, naturally pro- 1993). E. avium, E. saccharominimus, E. cecorum and E. duced by microorganisms, which vary in spectrum and mode of M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 3 activity, molecular structure and molecular mass, thermosta- addition, the bacteriocins produced by E. faecalis strains are bility, pH range of activity, and genetic determinants (Klaen- referred to as: hammer, 1993; De Vuyst and Vandamme, 1994; Nes et al., 1996; Nissen-Meyer and Nes, 1997; Moll et al., 1999; Ennahar Type 1: cytolysin (bacteriocin/hemolysin) from E. faecalis et al., 2000; Cleveland et al., 2001; McAuliffe et al., 2001; DS16 (Gilmore et al., 1994). This two-component lantibio- Riley and Wertz, 2002). tic displays both hemolytic and bacteriocin activity. Based on structural, physicochemical and molecular prop- Type 2: the cyclic peptide antibiotic AS-48 (enterocin AS- erties, bacteriocins from LAB can be subdivided into three 48) from E. faecalis S-48 (Martı´nez-Bueno et al., 1994). major classes (Klaenhammer, 1993; Nes et al., 1996). This compound is active towards both Gram-positive and Nonetheless, this classification is continuously being reviewed Gram-negative bacteria. In contrast, the identical enterocin 4 and it is evolving with the accumulation of knowledge and the produced by E. faecalis INIA 4 is only active towards appearance of new bacteriocins (Cleveland et al., 2001; Gram-positive bacteria (Joosten et al., 1996). Ennahar et al., 2000; Riley and Wertz, 2002; Rodrı´guez et Type 3: bacteriocin 31 from E. faecalis YI17 (Tomita et al., al., 2003). Class I bacteriocins are lantibiotics, i.e. small, 1996) with a narrow antibacterial spectrum. cationic, hydrophobic, and heat-stable peptides that contain Type 4: enterocin 1071A and enterocin 1071B from E. unusual amino acids (e.g. the thioether amino acids lanthionine faecalis BEF 1071 (Balla et al., 2000). These enterocins and/or 3-methyl-lanthionine) that are post-translationally present an activity spectrum narrower than Type 2 and formed. Class II bacteriocins are small, cationic, hydrophobic, broader than Type 3 enterocins produced by E. faecalis. heat-stable peptides that are not post-translationally modified, except for cleavage of a leader peptide from the prebacteriocin Enterocins, as most bacteriocins, have the cytoplasmic peptide. Within this class, three subclasses can be distin- membrane as their primary target. They form pores in the cell guished: subclass IIa or pediocin-like bacteriocins with a strong membrane, thereby depleting the transmembrane potential and/ antilisterial effect, possessing the consensus sequence YGNGV or the pH gradient, resulting in the leakage of indispensable in their N-terminus; subclass IIb or bacteriocins that require intracellular molecules (Cleveland et al., 2001). two polypeptide chains for full activity; and subclass IIc or bacteriocins that do not belong to the other subgroups. Class III 2.2. Citrate metabolism by enterococci bacteriocins are a group of large, hydrophilic, heat-labile proteins. Citrate is present in many raw materials that are used in food Since 1955, when the first bacteriocin-like substance within fermentations, such as fruit, vegetables, and milk, and it is also the group D streptococci was reported (Kjems, 1955), a large used as an additive for the production of fermented sausages. number of enterocins has been studied. Kra¨mer and Brandis The behavior of LAB may differ from one species to another, (1975) reported the anti-Listeria activity of the enterocin E1A and not all LAB are able to metabolize citrate. The ability to produced by E. faecium E1. Nowadays, the ability of metabolize citrate is invariably linked to endogenous plasmids enterococci to inhibit Listeria spp. is well known, which may that contain the gene encoding the transporter, which is be explained by the close phylogenetic relationship of responsible for citrate uptake from the medium. Since citrate enterococci and listeriae (Stackebrandt and Teuber, 1988; is a highly oxidized substrate, no reducing equivalents, such as Devriese and Pot, 1995). The first enterocin purified to NADH, are produced during its degradation, which results in homogeneity was the enterocin AS-48 produced by E. faecalis the formation of metabolic end products other than lactic acid. S-48 (Ga´lvez et al., 1989; Martı´nez-Bueno et al., 1994), which These include diacetyl, acetaldehyde, acetoin, and 2,3-butane- was defined as a cyclic peptide antibiotic. Since then, the diol, which have very distinct aroma properties and signifi- number of new enterocins characterised is increasing. Howev- cantly influence the quality of fermented foods (Hugenholtz, er, many of these molecules have not been purified to 1993). For instance, diacetyl determines the aromatic properties homogeneity. This is the case for the bacteriocins produced of fresh cheese, fermented milk, cream, and butter. The by E. faecalis E-1 (Bottone et al., 1971), E. faecium E1 breakdown of citrate also results in the production of carbon (Kra¨mer and Brandis, 1975), E. faecalis E23 (Nakagawa, dioxide, which can contribute to the texture of some fermented 1979), E. faecium (Harasawa et al., 1980), E. faecium S-34 dairy products. (Nakagawa and Matsuo, 1981), E. faecium 3(Kra¨mer et al., Most of the knowledge on the metabolic pathways involved 1983), E. faecium 25 (Reichelt et al., 1984), E. faecalis K4 in citrate metabolism has been derived from LAB of dairy (Ku¨hnen et al., 1985), E. faecalis DS16 (Ike et al., 1990), E. origin, although citrate itself does not always support their faecium 100 (Kato et al., 1993), E. faecalis 226 NWC (Villani growth. This claim is based on the inability of certain LAB to et al., 1993), E. faecium NA01 (Olasupo et al., 1994), E. grow in batch cultures to which excess citrate was added as the faecium 7C5 (Torri Tarelli et al., 1994), and E. faecium L1 sole energy source. Nevertheless, most LAB are able to co- (Lyon et al., 1995). metabolize citrate in the presence of another energy source, Enterocins are found within the class I, class IIa, class IIc, such as glucose or lactose. The metabolism of citric acid has and class III bacteriocins mentioned above (Table 1). Other been extensively studied and is well documented in strains of enterocins that at first were considered as new enterocins, but Lactococcus, Lactobacillus, and Leuconostoc (Drinan et al., were actually identical to existing ones, are listed in Table 2.In 1976; Cogan, 1987; Kennes et al., 1991; Starrenburg and 4 M.R. Foulquie´ 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 Referencea

Cyl LL E. faecalis DS16 MENLSVVPSFEELSVEEMEAIQGSGDVQAETTPVQ Gilmore et al., 1994 CAVAATAAASSAACGWVGGGIFTGVTVVVSLKHC

Cyl LS E. faecalis DS16 VLNKENQENYYSNKLELVGPSFEELSLEEMEAIQGQ Gilmore et al., 1994 SGDVQAETTPACFTIGLGVGALFSAKFC

Class IIa Producer strain MMb Amino acid sequence of the purified enterocin pIc Reference Enterocin A E. faecium CTC492 4833 TTHSGKYYGNGVYCTKNKCTVDWAKATTQ 9.0 Aymerich et al., 1996 CIAGMSIGGFLGGAIPGKC Enterocin CRL 35 E. faecium CRL 35 KYYGNGVTLNKXGXSVNXXXA... Farı´as 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 9.6 Eguchi et al., 2001 VKAYVNGFTKVLG

Class IIc Producer strain MM Amino acid sequence of the purified enterocin pI Reference Enterocin AS-48 E. faecalis S-48 7167 MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVQ 10.6 Martı´nez-Bueno GSGGLSLLAAAGRESIKAYLKKEIKKKGKRAVIAW 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 10.8 Ga´lvez et al., 1998; YGRYPWERPVA Sa´nchez-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 10.3 Balla et al., 2000; DRINRKKH 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 11.1 Yamamoto et al., 2003 WIARMWRT

Class III Producer strain MM Amino acid sequence of the purified enterocin pI Reference Enterolysin A E. faecalis LMG 2333 34501 ASNEWSWPLGKPYAGRYEEGQQFGNTAFNRGGTYQ 9.5 Nilsen et al., 2003 FHDGFDFGSAIYGNGSVYAVHDGKILYAGWDPVGGQ GSLGAFIVLQAGNTNVIYQEFSRNVGDIKVSTGQTVQ KKGQLIGKFTSSHLHLGMTKKEWRSAHSSWNKDDQ GTWFNPIPILQGGSTPTPPNPGPKNFTTNVRYGLRVQ LGGSWLPEVTNFNNTNDGFAGYPNRQHDMLYIKVDQ KGQMKYRVHTAQSGWLPWVSKGDKSDTVNGAAGMQ PGQAIDGVQLNYITPKGEKLSQAYYRSQTTKRSGWLQ KVSADNGSIPGLDSYAGIFGEPLDRLQIGISQSNPF 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; interest in citrate metabolism by Enterococcus strains has Kimoto et al., 1999; Goupry et al., 2000). In contrast, more probably arisen due to the fact that enterococci, in particular the limited and sporadic data concerning citrate metabolism by major species E. faecium and E. faecalis, frequently occur in Enterococcus strains are available (Coventry et al., 1978; large numbers in many cheeses, especially those manufactured Hagrass et al., 1991; Urdaneta et al., 1995; Freitas et al., 1999). in the Mediterranean area. Milk citrate catabolism by entero- Nevertheless, during the last few years more studies have cocci may explain, among other mechanisms, the role of focused on citrate metabolism by enterococci (Sarantinopoulos enterococci in the development of the distinctive organoleptic et al., 2001a,b, 2003; Rea and Cogan, 2003a,b). This increased properties of these cheeses. M.R. Foulquie´ 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 Morovsky´ et al., 2001 Enterocin CCM 4231 E. faecium CCM 4231 Foulquie´ 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 Foulquie´ Moreno et al., 2003a Enterocin RZS C5 E. faecium RZS C5 Foulquie´ 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 glucose, and in MRS broth containing glucose with increasing Gunsalus and Campbell (1944) showed that pH had a very citrate concentrations. This phenomenon was explained by significant effect on product formation from citrate in Rea and Cogan (2003a) who studied the factors affecting co- enterococci. Twenty-five years later, Devoyod (1969) pin- metabolism of citrate and glucose by growing and resting pointed that E. faecalis subsp. liquefaciens had the ability to cells of three E. faecalis and two E. faecium strains. It was metabolize citrate in the absence of carbohydrates. Since then suggested that the presence of glucose prevents citrate there appears to have been little work on citrate metabolism by utilization through catabolite repression, so that citrate can enterococci, until Hagrass et al. (1991) studied two strains of E. only be utilized when all of the glucose in the medium is faecalis, isolated from a fermented milk product. They showed consumed. The same authors suggested that catabolite that compounds, such as acetaldehyde and diacetyl, were repression by glucose and fructose occurs in E. faecalis produced via citrate metabolism. Raffe (1994) observed that E. strains, but this is not the case when galactose or sucrose are faecalis strains, grown in skim milk, could produce lactic acid used as energy sources (Rea and Cogan, 2003b). In contrast, from lactose, and acetic acid from citrate. During the same Sarantinopoulos et al. (2003) observed co-metabolism of period, Urdaneta et al. (1995) studied citrate metabolism in glucose and citrate for an E. faecium strain, grown in MRS three E. faecalis strains, grown in synthetic media, having broth, both under aerobic and anaerobic conditions and citrate as the sole energy source. All strains not only grew in uncontrolled pH. These results fully support the findings of those media, but they also produced acetate as the final Sarantinopoulos et al. (2001a) who, after an extensive product. Moreover, Freitas et al. (1999) concluded that E. screening of 129 E. faecalis, E. faecium and E. durans faecalis and E. faecium strains, which were grown in sheeps’ strains for general biochemical properties, showed that the and goats’ milk, produced relatively high amounts of lactic above set of strains exhibited strain-to-strain variation acid and lower amounts of formate and acetate, indicating concerning citrate metabolism, but with the majority of the indirectly that lactose and citrate were co-metabolised. E. faecalis strains able to utilize citrate. Recently, Sarantinopoulos et al. (2001b) showed that E. faecalis FAIR-E 229, a strain isolated from Cheddar cheese, 2.3. Proteolysis and lipolysis by enterococci was able to grow in skim milk, and co-metabolism of lactose and citrate took place. Moreover, when increasing initial 2.3.1. Proteolysis concentrations of citrate were used as the sole energy source, The degradation of casein plays a major role in the the main end products were acetate and formate, as a result of development of texture in cheese. In addition, some peptides citrate consumption. Surprisingly, the same strain did not contribute to the formation of flavour, whereas other, utilize citrate in MRS broth containing lactose instead of undesirable bitter-tasting peptides can lead to off-flavours. 6 M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24

Also, the secondary degradation of amino acids is considered concentrations inhibited it. The same author concluded that the to have a major impact on the flavour development in cheese. activity of an extracellular proteinase produced by the same Besides the indigenous milk proteolytic enzymes and rennin strain (S. faecalis subsp. liquefaciens 3/6) was reduced when used for protein coagulation, LAB cell wall-associated the strain was grown in milk that was processed at high proteinases and intracellular peptidases released after cell temperature (Hegazi, 1990b). This specific proteinase was able lysis in the curd are considered to play an important role in to hydrolyze casein, h-lactoglobulin and a-lactalbumin. The casein hydrolysis during cheese preparation (Wilkinson et al., activity of the enzyme was strongly reduced in the presence of 1994). EDTA and for that reason it was considered a metalloenzyme. Studies performed so far concerning proteolysis by LAB are At the same time, Garcia de Fernando et al. (1991a,b) mainly restricted to the genera Lactococcus and Lactobacillus examined in depth the extracellular proteinase production by E. (Pritchard and Coolbear, 1993; Sousa et al., 2001). The faecalis subsp. liquefaciens. The microorganism grew well and biochemical pathways, which lead to casein degradation and developed extracellular proteinase activity on proteinaceous peptide/amino acid transportation, are believed to be the same media. The molar mass of the enzyme has been estimated to be in the case of the other LAB, including the genus Enterococ- approximately 30 kDa by Sephadex gel filtration and approx- cus. However, only sporadic data exist in the literature imately 26 kDa by sodium-dodecyl sulphate-polyacrylamide regarding the proteolytic system of enterococci, in comparison gel electrophoresis (SDS-PAGE). The isoelectric point has with other LAB species. Even though there are no commonly been found to be 4.6 and maximum enzyme activity of the accepted laboratory methods in the literature for determining proteinase has been observed at pH 7.5 and 45 -C. The enzyme proteolytic and peptidolytic activities, such activities are hydrolyzed casein, bovine serum albumin, a-lactalbumin and generally low for Enterococcus strains, with the exception of h-lactoglobulin. Macedo et al. (2000) suggested that an E. E. faecalis strains (Dovat et al., 1970; Arizcun et al., 1997; faecium strain isolated from Serra cheese exhibited neglecting Macedo and Malcata, 1997; Suzzi et al., 2000; Andrighetto et aminopeptidolytic and endopeptidolytic activity in synthetic al., 2001). media, in comparison with strains belonging to the species of The very first work concerning the proteolytic system of L. paracasei, L. lactis, and Leuconostoc mesenteroides. enterococci was performed by Somkuti and Babel (1969). They Furthermore, Villani and Coppola (1994) examined the studied an extracellular proteinase, which was produced by an proteolytic activity of 24 E. faecium and 60 E. faecalis strains, E. faecalis var. liquefaciens strain. This proteinase was able to after growth in skim milk, at 37 -C for 6 h. All E. faecalis hydrolytically degrade casein in an intensive way and it was strains were much more proteolytic, in comparison with the E. less active against h-lactoglobulin and a-lactalbumin. In faecium strains. Quite recently, Andrighetto et al. (2001) another relevant study during that period, Wallace and Harmon showed that the majority of the 124 enterococcal strains (1970) examined an intracellular protease of an E. durans studied, isolated from traditional Italian cheeses, displayed strain. The protease was particularly active against casein and weak proteolytic activities in milk, but 30 of them belonging to h-lactoglobulin, but it was not able to hydrolyze bovine serum the E. faecalis species were more proteolytic. Finally, the same albumin. Furthermore, Dovat et al. (1970) studied the conclusion was drawn in another systematic study performed proteolytic activity of enterococci in milk and showed that by Sarantinopoulos et al. (2001a), who screened 129 E. they exhibited low proteolytic activity, with the exception of faecium, E. faecalis, and E. durans strains for biochemical strains belonging to the species E. faecalis var. liquefaciens. properties relevant to their technological performance. It was Almost 20 years later, Carrasco de Mendoza et al. (1989) found that all strains exhibited low extracellular proteolytic and determined in a more systematic study the extracellular peptidolytic activities, with the E. faecalis strains being proteolytic activity of 61 Enterococcus strains using sodium generally more active. caseinate as substrate. They suggested that the proteolytic activity was strain- and time-dependent. After 48 h of 2.3.2. Lipolysis incubation, strains belonging to E. faecalis species were more Lipids have a major effect on the flavour and texture of proteolytic, and this was more evident after 120 h. Further- cheese. They contribute to cheese flavour in three ways: (i) more, Wessels et al. (1990) examined 108 E. faecium, E. being a source of fatty acids, especially short-chain fatty acids, faecalis, and E. durans strains, isolated from various dairy whichmaybeconvertedtoothersapidandaromatic products. A large percentage of these strains of all species were compounds, especially methyl ketones and lactones; (ii) being able to grow at 7 -C, and more than half of them showed a cause of oxidation of fatty acids, leading to the formation of proteolytic activity at psychrotrophic temperatures. various unsaturated aldehydes that are strongly flavored and In another work, Hegazi (1990a) studied the proteolysis of S. thus cause a flavour defect referred to as oxidative rancidity; faecalis subsp. liquefaciens 3/6 in skim milk with some (iii) being solvents for sapid and aromatic compounds, additives. It was suggested that calcium lactate and some produced not only from lipids, but also from proteins and inorganic phosphate salts do not influence casein hydrolysis. In lactose. Milk fat hydrolysis during cheese manufacture and contrast, calcium carbonate, which is frequently used as an ripening is due to the endogenous milk lipase, the lipolytic additive in cheese manufacturing, resulted in a markedly enzymes of starter cultures and non-starter bacteria, lipases reduction of hydrolysis levels. Also, low NaCl concentrations from psychrotrophic bacteria, and–depending on the cheese (2%, w/v) positively influenced casein breakdown, while higher variety–exogenous enzyme preparations (El Soda et al., 1995). M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 7

LAB are generally considered to be weakly lipolytic, as durans strains were active against low-molecular-mass fatty compared with other groups of microorganisms. However, it is acids up to 4-nitrophenyl-caprylate (C8), while E. faecium was believed that lipolytic activity by LAB plays an important role active up to 4-nitrophenyl-stearate (C18). Differences between in the determination of the special aroma of many different the two species were also obvious for the post-electrophoretic cheeses. Certain LAB, which are used as bulk starter cultures detection of esterases. Moreover, Tsakalidou et al. (1994a) (Lactococcus and Lactobacillus), together with some other described the purification and characterization of an intracel- bacteria (Leuconostoc, Enterococcus, Pediococcus, and Mi- lular esterase of an E. faecium strain isolated from Feta cheese. crococcus), which can survive pasteurization and/or contam- The enzyme with a molecular mass of 45 kDa was optimally inate cheese during maturation, are believed to significantly active at 35 -C and pH 8.0. It could hydrolyze synthetic contribute to cheese fat hydrolysis (El Soda et al., 1995; substrates of low and medium molecular mass (C2 to C12) Collins et al., 2003). Concerning the lipolytic activity of with the highest activity against 4-nitrophenyl-butyrate (C4). enterococci, limited and often contradictory data exist in the Tsakalidou et al. (1994b) observed that enterococci gener- literature. ally exhibited higher esterolytic activities than other LAB The first work regarding the lipolytic and esterolytic species. In contrast, Villani and Coppola (1994) reported that activities of enterococci was performed by Lund (1965), who enterococci showed low lipolytic activity when grown in whole determined electrophoretically the presence of esterases in cell- milk. However, Tavaria and Malcata (1998) suggested that an free extracts of E. faecalis, E. faecium and E. durans strains. E. faecium strain isolated from Serra da Estrela cheese was able The electrophoretic pattern of the E. faecalis strains was to hydrolyze milk fat more extensively than a Lactococcus different compared to the patterns of the other two species. lactis subsp. lactis strain. In addition, Hemati et al. (1998) Moreover, E. faecalis strains exhibited higher activity, as reported that enterococci isolated from Domiati cheese determined on the basis of the intensity of the esterolytic bands. exhibited greater esterolytic activity in comparison with At the same time, Dovat et al. (1970) studied the lipolytic Lactobacillus strains. activity of 16 Enterococcus and 5 Streptococcus strains, after In another much more systematic study, Sarantinopoulos et they had grown in skim milk, milk cream and skim milk in the al. (2001a) showed that, among 129 enterococci, the majority presence of tributyrin, using as substrate Nile Blue Sulphate (90%) hydrolyzed all substrates from tributyrin (C4) to Agar. It was concluded that enterococci were more lipolytic. tristearin (C18) with a decreasing extent. In addition, The hydrolysis rate reduced with increasing molecular mass concerning esterases, all strains were active on the synthetic (tripropionin>tributyrin>tricaprin >tricaprylin), while triolein substrates used, from 4-nitrophenyl-acetate (C2) to -stearate was not hydrolyzed at all. In the same study, the volatile fatty (C18), with decreasing activity. Generally, E. faecalis strains acids produced were chromatographically determined, showing were the most lipolytic and esterolytic, followed by the E. that enterococci, and especially the E. durans strains, produced faecium and E. durans strains. Finally, while enterococci more acetic acid than the Streptococcus strains. isolated from Beyaz cheese showed a pronounced lipolytic A few years later, Martı´nez-Moreno (1976) showed that activity (Durlu-Ozkaya et al., 2001), the ones isolated from Enterococcus strains (E. durans, E. faecium, and E. faecalis) Semicotto Caprino cheese were not able to hydrolyse isolated from Manchego cheese, were active only against tributyrin, independent of the species (Suzzi et al., 2000). tributyrin and not against milk fat. Chander et al. (1979a,b) in two consecutive studies examined the influence of certain fatty 2.4. Enterococci as probiotics acids on the growth of and lipase production by E. faecalis, and they purified and characterized this lipase. It was observed that The definition of a probiotic has been evolving with the low-molecular-mass fatty acids (C3, C4, C6, and C10) increasing understanding of the mechanisms by which they enhanced lipase production, in contrast to high-molecular-mass influence human health (De Vuyst et al., 2004). The term was fatty acids (C12, C14, C16, C18, and C18:2) that inhibited first introduced to describe substances produced by one lipase production. The molar mass of this specific enzyme has microorganism (protozoa) that stimulate the growth of other been estimated to be approximately 21 kDa and the isoelectric microorganisms (Lilly and Stillwell, 1965). Nowadays, the point has been found to be 4.6. The maximum enzyme activity definition most commonly used is that of Fuller (1989), i.e. of the lipase has been observed at pH 7.5 and 40 -C. The ‘‘probiotics are live microbial feed supplements, which enzyme was able to hydrolyze tributyrin more than tricaproin, beneficially affect the host animal by improving its intestinal tricaprylin, and triolein. microbial balance’’. In 1992, the definition was expanded to After 10 years of silence on the lipolytic system of include food and non-food uses as well as the use of mono and enterococci, new studies have been published. Carrasco de mixed cultures (Havenaar and Huis in’t Veld, 1992). Hence, a Mendoza et al. (1992) concluded that the lipolytic activity of probiotic can be described as a preparation of or a product enterococci in milk was strain-dependent. Most of the strains containing viable, defined microorganisms in sufficient numb- examined exhibited low activity and only a few strains ers, which alter the microbiota (by implantation or coloniza- belonging to E. faecalis species were characterized as lipolytic. tion) in a compartment of the host and that exert beneficial In the same period, Tsakalidou et al. (1993) used synthetic health effects in this host (De Vuyst et al., 2004). They are substrates to detect photometrically and post-electrophoretical- applied in foods such as yogurt, fermented and non-fermented ly the esterolytic activities of E. faecium and E. durans. E. milks, infant formulas, and pharmaceutical preparations. 8 M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24

Probiotic consumption is reported to exert a myriad of in cholesterol levels in individuals who received GAIO, beneficial effects, including enhanced immune response, compared with that in control subjects at the end of the balancing of colonic microbiota, vaccine adjuvant effects, treatment (Richelsen et al., 1996; Sessions et al., 1997). In a reduction of fecal enzymes implicated in cancer initiation, recent work, the same fermented milk product GAIO was used treatment of diarrhea associated with travel and antibiotic to study the relationship of the E. faecium SF 68-containing therapy, control of rotavirus and Clostridium difficile-induced probiotic and a vancomycin treatment. It was concluded that E. colitis, and prevention of ulcers related to Helicobacter pylori. faecium SF 68 from the GAIO product was not found to Probiotics are also implicated in the reduction of serum acquire vancomycin resistance during the study period (Lund et cholesterol, the antagonism against food-borne pathogens and al., 2000). tooth decay organisms, the amelioration of lactose malabsorp- Rossi et al. (1999) used the strain E. faecium CRL 183 for tion symptoms as well as candidiasis and urinary tract the development of a novel fermented soymilk product with infections (Saavedra, 2001). A group of requirements have potential probiotic properties. E. faecium CRL 183 in been identified for a microorganism to be defined as an combination with Lactobacillus jugurti resulted in a 43% effective probiotic (Salminen et al., 1996). These include the decrease in cholesterol in vitro. Also, the probiotic strain E. ability to (a) adhere to cells; (b) exclude or reduce pathogenic faecium PR88 (E. faecium Fargo 688\ from Quest Interna- adherence; (c) persist and multiply; (d) produce acids, tional, Naarden, The Netherlands) was studied in a human hydrogen peroxide, and bacteriocins antagonistic to pathogen clinical trial. The consumption of this strain led to alleviation of growth; (e) be safe, noninvasive, noncarcinogenic, and the symptoms of irritable bowl syndrome in humans (Allen et nonpathogenic; and (f) coaggregate to form a normal balanced al., 1996). The strain was used for the production of a probiotic flora. Cheddar cheese. It did not affect cheese composition, but an Strains that are used as probiotics for man have been improvement of the flavour of the cheese was obtained isolated from the human gastrointestinal tract and usually (Gardiner et al., 1999). belong to species of the genera Lactobacillus and Bifidobac- Another probiotic product available on the market that terium. However, strains belonging to species of other LAB, contains an E. faecium strain is Walthers ECOFLOR (Walthers among them enterococci, have been used in the past as Health Care, Den Haag, The Netherlands). The producer claims probiotics too, such as E. faecium, E. faecalis, S. thermophilus, the efficacy of the E. faecium strain against diarrhea, its anti- L. lactis subsp. lactis, Leuconostoc mesenteroides, Propioni- carcinogenic effect, the production of enterocins active towards bacterium freudenreichii, Pediococcus acidilactici, Sporolac- L. monocytogenes, the possible decrease of the LDL-choles- tobacillus inulinus, E. coli, Bacillus cereus (Ftoyoi_), and the terol level, as well as its sensitivity to vancomycin and the yeast Saccharomyces cerevisiae (Fboulardii_)(Fuller, 1989; production of L(+) lactic acid (personal communication). O’Sullivan et al., 1992; Holzapfel et al., 1998). Even though However, the producer provides no scientific evidence. strains of E. faecium and E. faecalis species have been applied The use of enterococci as probiotics remains a controversial in human, probiotic supplements, E. faecalis strains have also issue. While the probiotic benefits of some strains are well been widely used as veterinary feed supplements. Since established, the emergence and the increased association of February 2004, 10 preparations (9 different strains of E. enterococci with human disease and multiple antibiotic faecium) are authorized as additives in feeding stuffs in the resistances (see below) have raised concern regarding their European Union (European Commission, 2004). use as probiotics. The fear that antimicrobial resistance genes A well-studied Enterococcus strain used as probiotic is E. or genes encoding virulence factors can be transferred to other faecium SF 68, which is produced in Switzerland (Cylactin\ bacteria in the gastrointestinal tract contributes to this LBC SF68 ME10, F. Hoffmann-La Roche Ltd., Basel, controversy (Franz et al., 2003). Switzerland). It has been proposed to be clinically effective in the prevention of antibiotic-associated diarrhea (Wunderlich 3. Pathogenesis of enterococci et al., 1989) and in the treatment of diarrhea in children (Bellomo et al., 1980). E. faecium SF 68 has been tested in Enterococci are among the most common nosocomial adults with acute diarrhea in two hospitals in Belgium pathogens and they have been implicated as an important (Buydens and Debeuckelaere, 1996). Although this type of cause of endocarditis, bacteremia, and infections of the urinary diarrhea was self-limited (>95% recovered by 6 days), the use tract, central nervous system, intra-abdominal and pelvic of the enterococci shortened the duration of diarrhea by 1–3 infections, as well as of multiple antibiotic resistances (Endtz days. E. faecium SF 68 has also been studied as a feed et al., 1999; Franz et al., 1999a). Several virulence factors have probiotic. E. faecium SF 68 used in a dry dog food been described (Jett et al., 1994) and the number of antibiotic- significantly enhanced cell-mediated and humoral immune resistant enterococci (ARE), especially vancomycin-resistant functions in dogs (Benyacoub et al., 2003). In Denmark, a enterococci (VRE), is increasing (Endtz et al., 1999). fermented milk product that contains E. faecium SF 68 (GAIO; MD Foods, Aarhus, Denmark) has been sold for several years 3.1. Cytolytic activity of enterococci because of its hypocholesterolemic effect on individuals (Agerbaek et al., 1995). However, two long-term studies (12 In 1949, Sherwood et al. reported on some h-hemolytic weeks and 6 months, respectively) failed to show any reduction group D streptococci that inhibited growth of other bacteria. In M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 9 the following years, other research groups observed the same polymyxins. The resistance to ampicillin (especially in E. phenomenon (for reviews, see Jett et al., 1994; Haas and faecium), tetracyclines, macrolides, aminoglycosides (high Gilmore, 1999). The evidence that bacteriocin and hemolytic level), chloramphenicol, trimethoprim/sulfamethozaxole, qui- activities were provided by a single compound came when both nolones, and streptogramins (in E. faecium and related species) activities were simultaneously lost after exposure to UV are acquired, as well as resistance towards glycopeptides irradiation but were simultaneously regained by reversion (vancomycin). VRE possess vanA, vanB, vanD, and vanE type (Brock and Davies, 1963). Granato and Jackson (1969, resistance genes (Klare et al., 2001). Ampicillin, vancomycin 1971a,b) found convincing data that E. faecalis hemolysin/ and gentamicin are the most clinically relevant antibiotics to bacteriocin was a natural bifunctional component. For simplic- cure infections with multiple ARE strains. It should be noted ity, the hemolysin/bacteriocin produced by E. faecalis has been that the extensive use of vancomycin has steadily raised the termed cytolysin (Gilmore et al., 1990). Amino acid analysis of number of VRE over the past two decades and therefore the the purified E. faecalis cytolysin revealed that it belongs to the percentage of invasive nosocomial enterococci displaying class I lantibiotics (Gilmore et al., 1994). Cytolysin is unique high-level vancomycin resistance (Endtz et al., 1999). How- among the lantibiotics in that it is able to lyse eukaryotic cells in ever, antibiotic resistance as such cannot explain the virulence addition to its bactericidal effect. As a class I lantibiotic, of enterococci. Unfortunately, VRE are also highly resistant to cytolysin forms pores in the cytoplasmic membrane of bacterial all standard anti-enterococcal drugs (Landman and Quale, target cells. Sequence and complementation analysis revealed a 1997), and, therefore, VRE constitute a serious risk group. gene cluster containing six genes that are required for cytolysin Dutka-Malen et al. (1995) developed a PCR assay to detect production (Gilmore et al., 1994; Saris et al., 1996). The two glycopeptide resistance genotypes. genes that encode the cytolysin subunits are designated cylLL Glycopeptide-resistant enterococci are phenotypically and (encoding for a peptide of 68 amino acids) and cylLS (encoding genotypically heterogeneous. Of the six different VRE pheno- for a peptide of 63 amino acids). Only the strains that are able to types known to date (Leclercq et al., 1988, 1992; Quintiliani et express and secrete both subunits are hemolytic (Booth et al., al., 1993; Perichon et al., 1997; Fines et al., 1999; McKessar et 1996). The cytolysin genes are located on the highly transmis- al., 2000), phenotypes VanA and VanB are of the highest sible pheromone-responsive conjugative plasmid pAD1 (Ike et clinical importance as they are most frequently observed in two al., 1990). Pheromones are small peptides (seven to eight amino predominant enterococcal species, E. faecalis and E. faecium acids) secreted by E. faecalis that promote conjugative transfer (Murray, 1997, 1998; Cetnikaya et al., 2000). VanA-type strains of plasmids between strains (Ike and Clewell, 1984). These display high-level inducible resistance to both vancomycin and peptides are chromosomally encoded and are referred to as teicoplanin, following the acquisition of transposon Tn1546 or pheromones because they elicit a specific mating response from closely related mobile genetic elements (Arthur et al., 1993). plasmid-carrying donor cells. Typically, multiple pheromones VanB-type strains display variable levels of inducible resistance are secreted simultaneously by a given E. faecalis strain. In to vancomycin only (Arthur et al., 1996). addition to pheromones, each pheromone-responsive plasmid ARE are widespread in food. They have been found in meat encodes a secreted peptide that acts as a competitive inhibitor of products, dairy products, and ready-to-eat foods, and even its corresponding pheromone (Jett et al., 1994). within enterococcal strains used as probiotics (Franz et al., Cytolysin is considered a virulence factor of E. faecalis 2001; Giraffa, 2002). Franz et al. (2001) found acquired strains in animal models (Ike et al., 1984; Huycke et al., 1991; resistance traits for a number of antibiotics in enterococci from Jett et al., 1992; Chow et al., 1993; Singh et al., 1998). food origin throughout Europe. However, in general these However, the role of this factor in enterococcal pathogenicity strains did not show resistance to the clinically relevant remains unclear. In a recent study, E. faecalis isolated from antibiotics. Giraffa (2002) emphasizes the role played by patients with endocarditis or bacteremia and from healthy ARE, and especially VRE, as possible natural food reservoirs volunteers were investigated for their ability to adhere to Int- in the dissemination of antibiotic-resistant traits in the environ- 407 and Girarti heart cell lines (Archimbaud et al., 2002). No ment. There could be a link between the use of some antibiotics link between the presence of some virulence factors such as in livestock and humans becoming colonised by ARE via the gelatinase, aggregation substances, and cytolysin, and the food chain. For safety reasons, a critical or important criterion to ability of the strains to adhere to these cells could be found. check is the absence of transferable antibiotic resistance in enterococci that could be used as co-cultures or starter cultures 3.2. Antibiotic resistance of enterococci in foods, since it is strain-specific (Franz et al., 2001; Vancanneyt et al., 2002; De Vuyst et al., 2003). Antibiotic resistance encompasses both natural (intrinsic) resistance and acquired (transferable) resistance. Enterococci 3.3. Virulence factors of enterococci possess a broad spectrum of antibiotic resistances within these two types (Klare et al., 2001). Examples of intrinsic resistance A virulence factor is an effector molecule that enhances the are vancomycin (Van C type) resistance in E. gallinarum, and ability of a microorganism to cause disease beyond that resistance towards streptogramins in E. faecalis, as well as intrinsic to the species background (Mundy et al., 2000). resistance to isoxazolylpenicillins, cephalosporins, monobac- Typical examples are aggregation substance, gelatinase, extra- tams, aminoglycosides (low level), lincosamides (mostly), and cellular superoxide, and extracellular surface protein. 10 M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24

3.3.1. Aggregation substance 3.4. Opsonophagocytic assay to assess the safety of potential Aggregation substance (Agg) is a pheromone-inducible enterococcal pathogens surface protein of E. faecalis, which promotes aggregate formation during bacterial conjugation (Clewell, 1993). As an Opsonophagocytic killing is used as an in vitro test to detect a important component of the bacterial pheromone-responsive protective immune response against bacterial pathogens (Pier et genetic exchange system, Agg mediates efficient enterococcal al., 1994), as all the antibodies that provide antibacterial donor–recipient contact to facilitate plasmid transfer (Clewell, immunity are opsonic. E. faecalis and E. faecium can posses a 1993). This trait may contribute to the pathogenesis of capsular polysaccharide that is the target for opsonic antibodies enterococcal infection through different mechanisms. The (Huebner et al., 1999). Hufnagel et al. (2003) compared one cells that express this trait form large aggregates in vivo. probiotic E. faecalis strain with a collection of clinical isolates However, it is unknown how this influences phagocytosis and and found that 89% of the clinical strains were less susceptible to subsequent damage of the vital functions of the organism. killing mediated by normal rabbit sera. The results showed a Also, Agg may bind on and present its cognate ligand to the strain-dependent susceptibility to opsonic killing. Moreover, surface of the organism, possibly resulting in superantigen opsonophagocytic killing is considered to be an important test to activity. Finally, Agg increases the hydrophobicity of the assess the safety of enterococcal strains (Hufnagel et al., 2003). enterococcal surface, which may induce localization of cholesterol to phagosomes, and prevent or delay fusion with 4. Enterococci in food lysosomal vesicles (Mundy et al., 2000). Studies on endo- carditis showed synergism between cytolysin and Agg. Up to Enterococci are widely distributed in the environment, now, Agg is exclusively found in E. faecalis strains; however, principally inhabiting the human and animal gastrointestinal its incidence among food isolates seems to be high (Eaton tract. E. faecalis is often the predominating Enterococcus and Gasson, 2001; Franz et al., 2001). species in the human bowel, although in some individuals and in some countries E. faecium outnumbers E. faecalis (Devriese 3.3.2. Gelatinase and Pot, 1995). However, Mundt (1986) demonstrated that the Gelatinase (Gel) is an extracellular metallo-endopeptidase common presence of E. faecalis in many food products is not involved in the hydrolysis of gelatin, collagen, haemoglobin, always related to direct faecal contamination. In 1992, the EU and other bioactive peptides (Su et al., 1991). The association established a maximum level for the presence of coliforms and between an enterococcal protease and virulence was first Escherichia coli, both considered as indicators of hygiene, suggested in 1975 by Gold et al., who found that a gelatin- while no limit was set for the enterococci (Anonymous, 1992). liquefying, human, oral E. faecalis isolate induced caries Furthermore, it has been shown that enterococci had little value formation in germ-free rats, while non-proteolytic strains did as hygiene indicators in the industrial processing of foods not. Su et al. (1991) sequenced the protease gene (gelE)inE. (Birollo et al., 2001). faecalis. Singh et al. (1998) demonstrated that Gel, which is Moreover, although E. faecalis, E. faecium, and E. durans commonly produced by nosocomial, fecal, and clinical are frequently isolated from human faeces, they are much less enterococcal isolates, is a virulence factor of enterococci, at prevalent in livestock, such as pigs, cattle, and sheep (Franz least for peritonitis in mice. The presence of Gel production et al., 1999a). Enterococci are not only associated with warm- among food E. faecalis strains is high (Eaton and Gasson, blooded animals, but they also occur in soil, surface waters, 2001; Franz et al., 2001). However, Eaton and Gasson (2001) and on plants, vegetables, and insects (Deibel and Silliker, demonstrated that even when the gel gene is present, a 1963; Mundt, 1986). The resistance of enterococci to negative phenotype can be found. None of the E. faecium pasteurization temperatures, and their adaptability to different strains involved in both studies produce Gel. substrates and growth conditions (low and high temperature, extreme pH, and salinity) implies that they can be found 3.3.3. Extracellular surface protein either in food products manufactured from raw materials Extracellular surface protein (Esp) was first described in a (milk or meat) and in heat-treated food products. This means clinical E. faecalis MMH594 isolate by Shankar et al. (1999). that these bacteria could withstand usual conditions of food The esp gene is unusually large consisting of 5622 nucleo- production. In addition, they can contaminate finished tides capable of encoding a primary translation product of products during food processing. Therefore, enterococci can 1873 amino acids, with a theoretical molecular mass of become an important part of the fermented food microbiota, approximately 202 kDa. Studies on the distribution of this especially in fermented cheeses and meats. surface protein revealed a significant enrichment in infection- The contribution of enterococci to the organoleptic proper- derived E. faecalis isolates (Shankar et al., 1999; Waar et al., ties of fermented food products and their ability to produce 2002). Esp is thought to play a role in adhesion and evasion bacteriocins (enterocins) are important characteristics for their of the immune response of the host. The incidence of Esp in application in food technology. In recent years, the reports food isolates differs for E. faecium and E. faecalis. While it about enterococci used as starter cultures or co-cultures is hardly found in E. faecium, the incidence in E. faecalis is (adjuncts) have increased considerably. The major part of this higher than expected. No explanation could be found up to kind of research has focused on the applicability of Entero- now (Eaton and Gasson, 2001; Franz et al., 2001). coccus strains, which harbor interesting biochemical and M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 11 technological properties, in cheese products and, to less extent, of animals, the milking equipment, and bulk storage tanks in meat products. However, the selection of enterococci to be (Gelsomino et al., 2001, 2002; Giraffa, 2002). involved in food processes is a difficult task, due to their Levels of enterococci in different cheese curds range from potential risk in human health (Vancanneyt et al., 2002; De 104 to 106 CFU gÀ 1 and in the fully ripened cheeses from 105 to Vuyst et al., 2003). 107 CFU gÀ 1 (Del Pozo et al., 1988; Litopolou-Tzanetaki, 1990; Litopoulou-Tzanetaki and Tzanetakis, 1992; Poullet et 4.1. Cheese products al., 1993; Kalogridou-Vassiliadou et al., 1994; Macedo et al., 1995; Centeno et al., 1996; Cuesta et al., 1996; Za´rate et al., 4.1.1. Presence of enterococci in cheese 1997; Franz et al., 1999a; Pe´rez Elortondo et al., 1999; Enterococci are associated with traditional European Xanthopoulos et al., 2000; Sarantinopoulos et al., 2001a; Franz cheeses manufactured in Mediterranean countries, such as et al., 2003; Giraffa, 2002; Manolopoulou et al., 2003), with E. Greece, Italy, Spain and Portugal, from raw or pasteurized faecium and E. faecalis being the dominant species. More goats’, ewes’, water-buffalos’ or bovine milk (Ordon˜ez et al., interestingly, in cheeses such as Manchego (Ordon˜ez et al., 1978; Trovatelli and Schliesser, 1987; Coppola et al., 1988; Del 1978), Mozzarella (Coppola et al., 1988), Kefalotyri (Litopo- Pozo et al., 1988; Litopolou-Tzanetaki, 1990; Litopoulou- lou-Tzanetaki, 1990), Picante de Beira Baixa (Freitas et al., Tzanetaki and Tzanetakis, 1992; Poullet et al., 1993; Kalo- 1995), Serra (Macedo et al., 1995), Cebreiro (Centeno et al., gridou-Vassiliadou et al., 1994; Torri Tarelli et al., 1994; 1996), Comte´(Bouton et al., 1998), Domiati (Hemati et al., Macedo et al., 1995; Centeno et al., 1996; Cuesta et al., 1996; 1998), and Kashar (Aran, 1998), enterococci are the predom- Za´rate et al., 1997; Aran, 1998; Bouton et al., 1998; Pe´rez inant microorganisms in the fully ripened product, while Elortondo et al., 1999; Xanthopoulos et al., 2000; Sarantino- according to the results of a 15-year survey, enterococci ranged poulos et al., 2002a; Manolopoulou et al., 2003). The from 104 to 106 CFU gÀ 1 in Emmental cheese and from 104 to prevalence of enterococci in milk and cheese has long been 107 CFU gÀ 1 in Appenzeller cheese (Teuber et al., 1996). The considered a result of unhygienic conditions during the European cheeses in which enterococci have been studied are production and processing of milk, such as direct contamina- compiled in Table 3. tion by animal and human feaces. Their presence has been Varying levels in different cheeses result from the cheese indirectly linked with contaminated water sources, the exterior 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 Comte´ 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 Arzu´ar Cow Centeno et al., 1995 Spanish Cebreiro Cow Centeno et al., 1996, 1999 Spanish Cebreiro Cow Mene´ndez 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 Simo´n Cow Garcı´a et al., 2002 Spanish Tenerife goat cheese Goat Za´rate et al., 1997 Spanish Tetilla Cow Mene´ndez et al., 2001 12 M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 and survival in the dairy environment (dependent on seasonal starters for the production of Cheddar cheese. They concluded temperature), as well as survival and growth under the that the cheese batches manufactured with the addition of E. particular conditions of cheese manufacture and ripening faecalis strains exhibited greater proteolytic degradation in (Litopolou-Tzanetaki, 1990; Litopoulou-Tzanetaki and Tzane- comparison to the cheese batches manufactured without takis, 1992; Macedo et al., 1995; Sarantinopoulos et al., enterococci or with the addition of E. durans strains. More 2001a). recently, when E. durans strains were used as co-cultures in the Although some authors have claimed in the past that high production of Feta cheese and a white-brined cheese, the levels of contaminating enterococci could lead to deterioration proteolytic index was higher in the experimental cheese than in of sensory properties in some cheeses (Thompson and Marth, the control. That was justified by the fact that the degree and 1986; Lo´pez-Diaz et al., 1995), other literature reports discuss the formation rate of non-casein nitrogen (NCN, pH 4.6 soluble the beneficial role of the presence of enterococci in cheese. nitrogen) and of phosphotungistic acid (PTA)-soluble nitrogen- Enterococci contribute to the ripening and aroma development continuously increased with ripening time (Litopolou-Tzane- of these products due to their proteolytic and esterolytic taki et al., 1993; Tzanetakis et al., 1995). An increased water- activities, as well as the production of diacetyl and other soluble nitrogen content and proteolytic index was also important volatile compounds (Jensen et al., 1975a,b; Ordon˜ez observed when E. faecalis strains were used as adjunct starters et al., 1978; Trovatelli and Schliesser, 1987; Centeno et al., in cheeses such as Cebreiro (Centeno et al., 1999), Hispa´nico 1996, 1999; Casalta and Zennaro, 1997; Franz et al., 1999a, (Garde et al., 1997; Oumer et al., 2001) and Venaco (Casalta 2003; Sarantinopoulos et al., 2001a,b). Furthermore, entero- and Zennaro, 1997). cocci seem to play an important role in improving flavor More recently, Centeno et al. (1999) examined the effects of development and quality, not only of cheese, but also of other E. faecalis in Cebreiro cheese manufacture. It has been traditional fermented foods, such as vegetables and sausages concluded that h-casein was broken down to a greater extent (Franz et al., 1999a; Giraffa, 2002). in the batches containing E. faecalis strains than in the control ones. Moreover, the highest level of as1-casein hydrolysis and 4.1.2. Applicability of enterococci as starter or adjunct cultures the highest ratio of peptide as1-I/as1-casein was obtained when in cheese E. faecalis strains were used. In all batches made with The presence of enterococci in high numbers in many enterococci, volatile free fatty acids, long-chain free fatty different types of cheeses, their potential contribution to the acids, and diacetyl and acetoin were higher than in the control organoleptic properties of fermented food products, and their batches. Increase of free fatty acids in cheese containing ability to produce bacteriocins (enterocins) are important enterococci was seen in Cheddar (Jensen et al., 1975a) and Feta characteristics to be considered by food processors for their cheese (Sarantinopoulos et al., 2002a) too. However, Centeno direct application in food manufacture. During the last few et al. (1999) recommended the use of moderate proteolytic (and years, the reports about enterococci proposed to be used as lipolytic) strains of E. faecalis to guarantee the quality of starter cultures or co-cultures (adjuncts) have considerably traditional Cebreiro cheese. increased. The aim of these studies was to elucidate the role that Finally, Sarantinopoulos et al. (2002a) studied in detail the enterococci play in cheese ripening. However, as enterococci effect of two E. faecium strains, as adjunct starter cultures, on have been recognized in recent years as major nosocomial the microbial, physicochemical and sensory characteristics of pathogens, one should carefully consider the potential virulence Feta cheese. Both enterococcal strains positively affected taste, factors of this group of microorganisms before use. aroma, color, and structure of the full-ripened cheeses, as well It has been proposed that enterococci can be included as part as the overall sensory profile. These results supported those of defined starter cultures for different European cheeses. The obtained by Litopolou-Tzanetaki et al. (1993) when E. durans effect of enterococci as starter cultures or co-cultures was strains were used for the production of Feta cheese. studied in cheeses, such as Cheddar (Dahlberg and Kosi- kowsky, 1948; Czulak and Hammond, 1956; Jensen et al., 4.1.3. Applicability of bacteriocinogenic enterococci and/or 1975a,b), Feta (Litopolou-Tzanetaki et al., 1993; Sarantino- their enterocins in cheese poulos et al., 2002a), water-buffalo Mozzarella (Coppola et al., There are many studies concerning the isolation and 1988; Parente et al., 1989; Villani and Coppola, 1994), Venaco characterization of bacteriocins (enterocins) produced by (Casalta and Zennaro, 1997), Cebreiro (Centeno et al., 1996, Enterococcus spp. Enterocins have been isolated from strains 1999), and Hispa´nico (Garde et al., 1997; Oumer et al., 2001). originating from different sources, including foods (cheese, In most of these studies, E. faecalis strains have been used, and meat, fish, and vegetables), animals, and humans. Table 4 only in the case of Feta cheese, strains belonging to the species shows a compilation of bacteriocinogenic Enterococcus strains of E. faecium (Sarantinopoulos et al., 2002a) and E. durans (Bac+) from different origins that have been studied. The (Litopolou-Tzanetaki et al., 1993) have been tested. Also, the enterococcal bacteriocins are usually active towards foodborne British FAdvisory Committee on Novel Foods and Processes_ pathogens such as Listeria spp. and Clostridium spp. (Ben (ACNFP) accepted the use of E. faecium strain K77D as a Embarek et al., 1994; Farı´as et al., 1994; Vlaemynck et al., starter culture in fermented dairy products (ACNFP, 1996). 1994; Giraffa et al., 1994, 1995a,b; Maisnier-Patin et al., 1996; In an early, comparative study, Jensen et al. (1975b) used Vlaemynck, 1996; Cintas et al., 1998a; Bennik et al., 1999; two E. faecalis strains and two E. durans strains as adjunct Mendoza et al., 1999). Activity towards Gram-negative M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 13

Table 4 (Table 5) and/or the corresponding enterocins produced (Table Origin of bacteriocinogenic Enterococcus strains 6) has been studied in a wide variety of fermented food Source Strain(s) Reference products (mainly in cheese products) at small, pilot-scale Dairy products E. faecium DPC1146 Parente and Hill, 1992a conditions. However, the functionality of such strains at large- E. faecium 7C5 Torri Tarelli et al., 1994 scale, industrial, food processing conditions is still unknown E. faecium RZS C5 Vlaemynck et al., 1994 and certainly needs to be thoroughly examined in the near E. faecalis INIA 4 Joosten et al., 1995 E. faecium CRL 35 Farı´as et al., 1996 future. E. faecium WHE 81 Ennahar et al., 1998 Reports on the optimization of enterocin production and Screening for Bac+ strains Carnio et al., 1999 growth of enterococci during batch fermentation are scarce. Screening for enterocin Rodrı´guez et al., 2000 Nevertheless, kinetic studies of batch fermentations can be AS-48 producer strains used to analyze the relation between bacteriocin production, E. faecalis TAB 28 Rodrı´guez et al., 2000 Screening for Bac+ strains Elotmani et al., 2002 growth of the producer organism, and the microbial environ- E. faecalis FAIR-E 309 Franz et al., 2002 ment. Such studies have indicated that the bacteriocin E. faecium FAIR E-198 Sarantinopoulos et al., 2002b production by E. faecium RZS C5 follows a growth- Screening for Bac+ strains Saavedra et al., 2003 associated process and is limited to the early growth phase Fermented E. faecium L1 Lyon et al., 1995 because of a shut off mechanism (Leroy and De Vuyst, 2002; sausages E. faecium CTC492 Aymerich et al., 1996 E. faecalis EFS2 Maisnier-Patin et al., 1996 Leroy et al., 2003a). Moreover, they indicated that the E. faecium T136 Casaus et al., 1997 kinetics of E. faecium RZS C5 are affected by the pH and E. faecium P13 Cintas et al., 1997 the temperature, and the concentration of sugar, complex E. faecium L50 Cintas et al., 1995, 1998b nutrients, and sodium chloride (Leroy and De Vuyst, 2002; E. faecium AA13 Herranz et al., 1999 Leroy et al., 2003b). Another very well studied bacteriocin is E. faecium G16 Herranz et al., 1999 E. faecium P21 Herranz et al., 2001 enterocin 1146 from E. faecium DPC 1146 (Parente and Hill, E. casseliflavus IM 416K1 Sabia et al., 2002 1992a,b,c; Parente and Ricciardi, 1994a,b; Parente et al., Other fermented E. faecium N15 Losteinkit et al., 2001 1997; Foulquie´ Moreno et al., 2003b). Enterocin 1146 has a foods E. faecium B1 Moreno et al., 2002 rapid bactericidal effect on L. monocytogenes in buffer E. faecium B2 Moreno et al., 2002 systems, broth and milk, and it is stable during the ripening Screening for Bac+ strains Onda et al., 2002 Screening for Bac+ strains Yamamoto et al., 2003 period of Cheddar cheese, as it is the case for E. faecium Fish Screening for Bac+ strains Ben Embarek et al., 1994 FAIR-E 171 (Foulquie´ Moreno et al., 2003b). As enterocin Vegetables E. faecalis 226 Villani et al., 1993 production by E. faecium FAIR-E 171 takes place from the E. faecium BFE 900 Franz et al., 1996 beginning of the cheese production and is stable during the E. mundtii ATO6 Bennik et al., 1998 whole cheese-ripening phase, both early and late contamina- E. faecium 6T1a Floriano et al., 1998 Silage Screening for Bac+ Kato et al., 1994 tion of the milk or cheese by Listeria spp. can be combated E. faecium strains with stable, in situ enterocin production. E. faecium RZS C13 Vlaemynck et al., 1994 Some successful examples of in situ enterocin production E. faecium NIAI 157 Ohmomo et al., 2000 during cheese making have been described and the stability E. faecalis K-4 Eguchi et al., 2001 of certain enterocins during cheese ripening has been Veterinary E. faecium CCM 4231 Laukova´ et al., 1993 E. faecium BC25 Morovsky´ et al., 1998 reported in Taleggio, Manchego, and Cheddar cheese E. faecium J96 Audisio et al., 1999 (Giraffa et al., 1994, 1995b; Giraffa and Carminati, 1997; E. faecalis BFE 1071 Balla et al., 2000 Nu´n˜ez et al., 1997; Foulquie´ Moreno et al., 2003b). Giraffa Screening for Bac+ strains du Toit et al., 2000 et al. (1994) showed that enterococci are suitable to produce E. gallinarum 012 Jennes et al., 2000 bacteriocins in milk, in the presence of rennet, during an Water E. faecalis EJ97 Ga´lvez et al., 1998 Human origin E. faecalis S-48 Ga´lvez et al., 1986 incubation that simulated the first 55 h of Taleggio cheese E. faecium YI717 Tomita et al., 1996 making. Nu´n˜ez et al. (1997) concluded that E. faecalis INIA Screening for Bac+ strains del Campo et al., 2001 4 is able to produce its enterocin in competition with the E. faecium RC714 del Campo et al., 2001 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 bacteria such as E. coli (Ga´lvez et al., 1989; Tomita et al., Taleggio cheese manufacture and ripening, Giraffa et al. 1997) and Vibrio cholerae (Simonetta et al., 1997) has been (1995b) showed that bacteriocin production by E. faecium shown as well. Furthermore, Wachsman et al. (1999) reported 7C5 starts during whey drainage and generally reaches a on the antiviral activity of enterocin CRL35. maximum amount just after cheese salting. The stability of For the above-mentioned reasons the use of bacteriocin- the enterocin during ripening is not affected, although the producing enterococci as starter cultures, co-cultures (adjunct heterogeneous microbiota present in raw milk, as well as the cultures) or protective cultures, as well as the direct addition of enzymatic activity of rennet, are capable to inactivate the the enterocins in food fermentation processes, is a point of inhibitor. Furthermore, E. faecium 7C5 inhibits the growth of great interest amongst fermentation technologists. As a L. monocytogenes Ohio on the surface of Taleggio cheese consequence, the applicability of such enterococci cultures (Giraffa and Carminati, 1997). 14 M.R. Foulquie´ 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 Nu´n˜ez 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 Laukova´ 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 Rodrı´guez et al., 2001 E. faecium RZS C5 Enterocin RZS C5 Cheddar cheese production Foulquie´ Moreno et al., 2003b E. faecium DPC 1146 Enterocin DPC 1146 Cheddar cheese production Foulquie´ 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 4.2. Meat products under the conditions resembling Feta cheese manufacture, enterocin production was low or negligible. Moreover and 4.2.1. Presence of enterococci in meat in contrast with the enterocin 1146, no enterocin activity The presence of enterococci in the gastrointestinal tract of was detected when the same strain was used as an adjunct animals may lead to contamination of meat at the time of starter culture for Feta cheese production (Sarantinopoulos slaughtering. In a study of enterococci from raw meat products, et al., 2002b). The latter phenomenon can be attributed to E. faecalis was the predominant isolate from beef and pork cuts the fact that enterocin production in milk using enterococci (Stiles et al., 1978). E. faecium was also frequently isolated as co-cultures can be lower than when enterococci are used from Bologna, a processed pack sausage (Stiles et al., 1978). as pure cultures (Giraffa et al., 1995a,b; Sarantinopoulos et Pig carcasses from three different slaughtering plants contained al., 2002b; Foulquie´ Moreno et al., 2003b). Another mean log counts of 104–108 enterococci per 100 cm2 of explanation could be that the enterocin is not produced at carcass surface. E. faecium and E. faecalis were the predom- all under the specific conditions of this technology or that it inant species isolated (Knudtson and Hartman, 1993). E. is produced but can not be detected due to adsorption on faecalis predominated the Gram-positive cocci isolated from the cheese matrix. This phenomenon has been reported chicken samples collected at poultry abattoirs. Enterococci before (Sulzer and Busse, 1991). Farı´as et al. (1999) were consistently isolated from beef, poultry, and pig carcasses, studied the effect of enterocin CRL35 used as additive or from fresh meat in studies of antibiotic ARE (Franz et al., during goat cheese making contaminated with Listeria. 2003). Although enterocin CRL35 could not be detected during Besides raw meats, enterococci are also associated with ripening, suppression of Listeria took place during the processed meats. Heating of processed meats during production whole period of ripening. may confer a selective advantage to enterococci because these bacteria are among the most thermotolerant of the non- sporulating bacteria. After surviving the heat-processing step, Table 6 both E. faecalis and E. faecium have been implicated in Applications of enterocins as additives spoilage of cured meat products, such as canned hams and Enterocin Application Reference chub-packed luncheon meats (Magnus et al., 1986, 1988). This Enterocin 226 NWC Mozzarella cheese Villani et al., 1993 is especially true where recontamination with competing Enterocin 4 A model dairy system Rodrı´guez et al., 1997 bacteria is prevented, i.e. when products are heated after Enterocin CCM 4231 Cattle slurry environment Laukova´ et al., 1998 Enterocin CCM 4231 Soy milk Laukova´ and Czikkova, packaging in cans or in impermeable plastic films. The heat 1999 resistance of enterococci in these products is influenced by Enterocin CCM 4231 Dry fermented Horna´d Laukova´ et al., 1999 components, such as salt, nitrite, and meat tissue (Bell and salami DeLacey, 1984; Magnus et al., 1986, 1988; Franz et al., Enterocin CCM 4231 Bryndza, a traditional Laukova´ and Czikkova´, 1999a). To prevent spoilage of the processed meats by Slovak dairy product 2001 from sheep milk enterococci, it was suggested that initial contamination by Enterocin CRL 35 Goat cheese making Farı´as et al., 1999 these microorganisms should be kept to a minimum, and that Enterocin CRL 35 Meat system Vignolo et al., 2000 adequate heat processing should be based on D-values of the Enterocin CTC 492 Meat products Aymerich et al., 2000b most heat-resistant enterococci isolated from raw materials Enterocin CTC 492 Cooked pork Aymerich et al., 2002 (Magnus et al., 1986). M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 15

Enterococci have also been isolated from some types of effect on the inhibition of Listeria growth (Aymerich et al., fermented sausages. Salami and Landja¨ger were shown to 2000a). In contrast, the combination of both E. faecium CTC contain enterococci at numbers ranging from 100 to 2.6Â105 492 and its enterocins can prevent ropiness in sliced cooked CFU gÀ 1 (Teuber et al., 1996). Enterococci were isolated from ham (Aymerich et al., 2002). dry fermented sausages such as Chorizo (Casaus et al., 1997; Furthermore, the antilisterial efficiency of enterocins Cintas et al., 1997) and Espetec (Aymerich et al., 1996), both CRL35 (produced by E. faecium CRL35) has been tested in produced in Spain. Moreover, in German and Italian fermented broth and a meat system against L. monocytogenes and L. sausages, Marchesino et al. (1992) reported concentrations of innocua (Vignolo et al., 2000). Both Listeria species initially enterococci from 103 to 105 CFU gÀ 1 at the end of the ripening decreased in viable counts followed by regrowth of the process in sausages manufactured both with and without starter survivors after 1 h in the presence of the enterocins. The cultures. organoleptic properties of the meat model system were not In Mediterranean countries, many traditional and artisan affected. Finally, the effectiveness of enterocin CCM 4231 in fermented meat products are manufactured mostly by small controlling L. monocytogenes contamination of dry fermented companies, farms, or local butchers. Usually, they are low-acid Horna´d salami has been examined (Laukova´ et al., 1999). The products with a pH higher than standard sausages (pH>5.3). In enterocin addition resulted in the reduction of L. monocyto- these types of products, the fermentation is carried out without genes by 1.67 logs when compared to the control immediately the use of starter cultures, relying on the endogenous flora to after the addition of bacteriocin. Although on the second day keep the traditional organoleptic qualities. As a consequence, the growth of the pathogen in the experimental sample reached the natural microbiota is constituted by a mixture of species of 3.38 log CFU gÀ 1, a difference of 1.72 log was found, in LAB including enterococci and lactobacilli, as well as comparison with the control. After one week of ripening the L. coagulase-negative staphylococci and yeasts. These products monocytogenes count in the control reached 107 CFU gÀ 1, have a long history of safe consumption by humans. In a while in the experimental sample the count was 104 CFU gÀ 1, survey of 31 naturally fermented sausages, purchased at several a difference that was maintained after 2 and 3 weeks of supermarkets in the northeast of Spain, the number of ripening. enterococci ranged from 1.3 to 4.48 log CFU gÀ 1,the As stated before and as found also in many other application population of total LAB being from 7.12 to 9.07 log CFU studies, it has been shown that there are limitations to the gÀ 1 (Hugas et al., 2003). usefulness of some enterocins as antilisterial agents, as full The biochemical activities of enterococci in a sausage suppression of the pathogen is rarely achieved in fermented matrix have not been studied as in the case of dairy products. food systems (e.g. cheeses). The optimal use of bacteriocins for They might contribute to sausage aromatisation by their inhibiting the growth of Listeria and other foodborne patho- glycolytic, proteolytic, and lipolytic activities. Furthermore, gens or food spoilage microorganisms requires detailed metmyoglobin reduction activity has been described for meat knowledge of their activity against sensitive strains and the enterococci (Hugas et al., 2003). factors that may limit their effectiveness. These factors can be revealed through a thorough examination of the foods’ 4.2.2. Applicability of bacteriocinogenic enterococci and/or structure and composition and their interaction with bacter- their enterocins in meat products iocins. As a consequence, there is a great need to maximize the Even though there are many bacteriocin-producing entero- amount of the bioavailable enterocin during the food fermen- coccal strains isolated from various fermented, meat products tation process, by selecting enterococcal strains that are (Table 4), only a very limited number of studies has been characterized by a naturally high bacteriocin production conducted aimed at the direct application of such strains in the capacity, or, much more importantly, which are well adapted production of meat products (Tables 5 and 6). Very recently, to the food environment in which they are to be used some attempts have been made using E. faecium strains as (Sarantinopoulos et al., 2002b). adjunct starter cultures or their enterocins for the manufacture of fermented, meat products. 4.3. Vegetables and olives E. faecium RZS C13 and E. faecium CCM 4231 were used as starter cultures for the production of Spanish-style Enterococci commonly occur in large numbers in vegeta- dry, fermented sausages (Callewaert et al., 2000). The bles, olives, and plant material (Mundt, 1963; Ferna´ndez-Diaz, Enterococcus strains were partially competitive during meat 1983; Franz et al., 1999a; Giraffa, 2002; Ben Omar et al., fermentation and strongly inhibited the growth of Listeria 2004). However, literature data concerning the isolation and spp. As no production of off-flavours was detected, the characterization of enterococci from such products is scarce. enterococci might be suitable for addition to meat as co- Enterococci have been isolated from Spanish-style green olive cultures to improve food safety. Aymerich et al. (2000b) fermentations (Ferna´ndez-Diaz, 1983; Floriano et al., 1998; de applied the enterocins A and B produced by E. faecium CTC Castro et al., 2002), in which E. faecalis and E. faecium are 492 as semi-purified food additives in different meat frequent contaminants. In a very recent study, Ben Omar et al. products. An inhibitory effect was observed on the growth (2004) studied the functional properties, the virulence char- of Listeria. However, the addition of the producer strain as acteristics, and the antibiotic resistance of fifty enterococcal the sole starter culture in dry fermented sausages had no isolates from different Spanish food samples. Three of them 16 M.R. Foulquie´ Moreno et al. / International Journal of Food Microbiology 106 (2006) 1–24 were isolated from olives and belonged to the species E. accept enterococci as other LAB as an important player in faecium. Moreover, it was found that the viable counts of certain food products. enterococci from olives obtained on Slanetz and Bartley medium was 2.2Â103 CFU gÀ 1. Acknowledgments In another very recent study, Randazzo et al. (2004) isolated a total number of 52 LAB from naturally fermented green This work was carried out in the framework of the FAIR- olives collected from different areas of Sicily. Even though the CT97-3078 project ‘‘Enterococci in Food Fermentations: majority of these strains belonged to the genus of Lactobacil- Functional and Safety Aspects’’. MRFM was recipient of a lus, four of them were identified as Enterococcus spp. on the Marie Curie Fellowship (grant FAIR-CT97-5013). PS was basis of phenotypic and biochemical traits. Characterization supported by the State Scholarships Foundation of Greece using restriction fragment length polymorphism (RFLP) (IKY-IDrima Kratikon Ypotrofion). LDV acknowledges the analysis of 16S rDNA revealed that these four strains belonged financial support from the Research Council of the VUB and to the species E. faecium, E. casseliflavus, and E. hirae. To our the Fund for Scientific Research-Flanders. knowledge there is only one study dealing with the role of enterococci during the fermentation of table olives (de Castro et References al., 2002). In this study, strain Enterococcus casseliflavus cc45, isolated from the fermentation brine, in combination with strain ACNFP, 1996. Report on Enterococcus faecium, strain K77D. MAFF Advisory Lactobacillus pentosus CECT 5138 of the same origin, were Committee on Novel Foods and Processes, Report, Ergon House c/o Nobel used as starter cultures in Spanish-style green olive fermenta- House, 17 Smith Square, London SW1 3JR, United Kingdom. Agerbaek, M., Gerdes, L.U., Richelsen, B., 1995. Hypocholesteroleamic effect tion. The best results were attained when cultures were used of a new fermented milk product in healthy middle-aged men. European sequentially, enterococci at day 1 and lactobacilli at day 2. This Journal of Clinical Nutrition 49, 346–352. type of inoculation produced a quicker brine acidification, a Allen, W.D., Linggood, M.A., Porter, P., 1996. Enterococcus organisms and greater consumption of carbohydrates, and a rapid pH decrease. their use as probiotics in alleviating irritable bowel syndrome symptoms. The diversity of vegetable products available on the European Patent 0508701 (B1). Andrighetto, C., Knijff, E., Lombardi, A., Torriani, S., Vancanneyt, M., European market is increasing, and minimally processed, Kersters, K., Swings, J., Dellaglio, F., 2001. Phenotypic and genetic ready-to-eat type products are popular among consumers. As diversity of enterococci isolated from Italian cheeses. 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