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Journal of Applied Microbiology 2004, 97, 621628

doi:10.1111/j.1365-2672.2004.02347.x

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese
` re2 T. Ghrairi1,2, M. Manai1, J.M. Berjeaud2 and J. Fre
1 2

culaire, Faculte des Sciences de Tunis, Campus universitaire, Tunis, Tunisia, and Laboratoire de Biochimie et Biologie Mole e CNRS-UMR 6008, IBMIG-PBS, Faculte des Sciences de Poitiers, Poitiers, France Laboratoire de Microbiologie Fondamentale et Applique

2003/0456: received 2 June 2003, revised 20 April 2004 and accepted 4 May 2004

ABSTRACT
` R E . 2004. T. GHRAIRI, M. MANAI, J.M. BERJEAUD AND J. FRE

Aims: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great signicance for the dairy industry to improve food safety. Methods and Results: Six-hundred strains of LAB isolated from rigouta, a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identied as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the puried antibacterial compound. Polymerase chain reaction experiments using nisin gene-specic primers conrmed the presence of nisin operon. Plasmid proles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identied, at molecular level, as Enterococcus faecalis. The puried bacteriocin produced by this strain showed a molecular mass of 10 20133 085 Da. This new member of class III bacteriocins was termed enterocin MMT05. Conclusions: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or benecial role in foods. Signicance and Impact of the Study: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety. Keywords: bacteriocin, enterocin, Enterococcus faecalis, Lactococcus lactis, nisin Z.

INTRODUCTION Lactic acid bacteria (LAB) were used extensively as starter cultures in food fermentation. They contribute to the organoleptic properties of the nal product, as well as its preservation and microbial safety. Their antimicrobial activity is mainly because of the production of lactic acid. In addition to organic acids, LAB can produce a range of other antimicrobial metabolites such as ethanol, diacetyl and hydrogen peroxide (Stiles and Holzapfel 1997; Ross et al.
re, Universite de Poitiers, IBMIG, 40 av du recteur Correspondence to: J. Fre Pineau, 86022 Poitiers, France (e-mail: jacques.frere@univ-poitiers.fr); M. Manai, de Tunis El Manar, Faculte des Sciences de Tunis, Campus El Manar, Universite 2092 Tunis, Tunisia (e-mail: mohamedmanai@yahoo.fr).

2002). Antimicrobial activity could also be associated with ribosomally synthesized proteinaceous inhibitors collectively known as bacteriocins. These antibacterial peptides are of great interest in food industry as natural preservatives and possible substitutes for chemical preservation (Abee et al. 1995; Cleveland et al. 2001; Ross et al. 2002). Bacteriocins produced by LAB have been classied on the basis of their biochemical characteristics in three groups (Klaenhammer 1993; Nes et al. 1996). The previously proposed class IV, consisting of bacteriocins that form large complexes with macromolecules (Klaenhammer 1993), is probably because of artefacts or partial purications (Cleveland et al. 2001). Class I bacteriocins, termed lantibiotics, are small and heat-stable peptides that contain thioether

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amino acids, like lanthionine. The model of this group, nisin, is probably the most well-known and studied bacteriocins produced by LAB (Cleveland et al. 2001; Ross et al. 2002). Class II contains small heat-stable, nonmodied peptides (Nes and Holo 2000) which are subdivided into Class IIa, or antilisterial peptides, that contains the recently described lactococcin MMFII (Ferchichi et al. 2001) and sakacin G (Simon et al. 2002) and Class IIb, consisting in the association of two different peptides for full activity, such as lactococcin G (Nissen-Meyer et al. 1992) or lacticin F (Muriana and Klaenhammer 1991). Class III contains large heat-labile proteins. This group is not well documented. Only few large bacteriocins produced by LAB are described at the molecular level, such as helveticin J produced by Lactobacillus helveticus 481 (Joerger and Klaenhammer 1990) or enterolysin A from Enterococcus faecalis LMG 2333 (Genbank accession number AF249740). Nevertheless, some of the incompletely described antibacterial compounds could be suspected to be class III bacteriocins, although not yet classied, e.g. helveticin V1829 from Lact. helveticus 1829 (Vaughan et al. 1992), the antimicrobial substance from Lact. helveticus CNRZ450 strain (Thompson et al. 1996) or the recently reported enterocin R69 from Ent. faecalis R69 (Elotmani et al. 2002). Nisin is produced by numerous Lactococcus lactis strains and has been approved in over 48 countries (E234 additive for European countries) for many applications in a large range of food products, particularly in cheese manufacturing (Ross et al. 2002). Two naturally occurring nisin variants namely nisin A and nisin Z that differ in a single amino acid residue but showing similar activities have been described. Nisin A possesses a histidine and nisin Z, an asparagine at position 27 (Mulders et al. 1991). Nisin kills sensitive strains by pore formation in the membrane of sensitive bacteria and is active at the nanomolar level by targeting the lipid-bound cell wall precursor lipid II as a docking molecule (Breukink et al. 1999; Wiedemann et al. 2001). Generally, bacteriocins exhibit inhibitory activity towards bacteria closely related to their producing strain (Nes et al. 1996) consequently, some L. lactis starter strains are inhibited by nisin. Nevertheless, producer strains are protected from their own bacteriocin by expressing a specic immunity protein. Thus, starter blends designed with nisin-producers associated to insensitive strains with appropriate technological properties could be of interest, particularly in cheese manufacturing (Roberts and Zottola 1993). Tunisian traditional dairy products produced with poor hygienic practices, compared with North European industrial dairy technology, suffer of inconsistent quality presentation and reduced shelf life. In such media, bacteria possessing advantages for environmental competition, such as bacteriocins production, could be isolated. In a previous report, we described the isolation from rigouta cheese and

subsequent characterization of the bacteriocinogenic L. lactis MMFII producing lactococcin MMFII, a class IIa bacteriocin (Ferchichi et al. 2001). Herein, we describe the partial characterization of antilisterial compounds produced by eight LAB selected from over 600 LAB isolated from Tunisian traditional cheeses.

MATERIALS AND METHODS Strains, media, culture conditions and chemicals Bacterial strains used in this study are listed in Table 1. All media were supplied by Difco (Detroit, MI, USA). Lactococcal strains were grown on medium M17 supplemented with 05% (w/v) lactose (LM17) or sucrose (SM17) at 30C for 24 h. Skimmed milk was prepared from skimmed milk powder as recommended by the supplier gilait, Les Jonchets, France). Enterococcal, Listeria and (Re Hafnia strains were grown on BHI for 24 h at 37C. Leuconostoc, Pediococcus and Lactobacillus strains were grown on MRS medium at 30C for 24 h. Escherichia coli and Serratia strains were grown on LB medium at 37C for 24 h. Brochotrix sp. was grown on APT medium for 24 h at 20C. Strains were maintained in their broths with 15% (v/v)
Table 1 Bacterial strains used in this study Strains L. lactis subsp. lactis ATCC11454* L. lactis subsp. cremorisATCC11603 Lact. casei DSM20011 Lact. delbruekii DSM20081 Lact. sakei 2525 Ent. faecium ENSAIA631 Ent. faecalis JH2-2 L. ivanovii BUG496 L. monocytogenes EGDe Leuc. mesenteroides Y105 Ped. acidilactici 1521 Serratia sp. Brochotrix sp. Hafnia sp. E. coli XL1-blue Reference or source ATCC ATCC DSM DSM Guyonnet et al. (2000) ENSAIA D. Clewell (University of Michigan) Institut Pasteur (Paris) Institut Pasteur (Paris) chard et al. (1992) He Guyonnet et al. (2000) Rhodia Food Rhodia Food Rhodia Food LMFA

ATCC, American Type Culture Collection; ENSAIA, Ecole Nationale dAgronomie et des Industries Agroalimentaire (Nancy, France); DSM, Deutche Sammlung von Mikroorganismen und Zellkulturen; e LMFA, Laboratoire de Microbiologie Fondamentale et Applique (Poitiers, France). *Producer of nisin A. Producer of sakacin P. Producer of mesentericin Y105 and mesentericin B105. Producer of pediocin PA-1. Strains isolated from food.

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glycerol at )20C. Acetonitrile, high-performance liquid chromatography (HPLC) grade, was supplied by Carlo Erba (Val de Reule, France). Other chemicals were provided by Sigma-Aldrich (St Louis, MO, USA). Detection of bacteriocin activity LAB were isolated from rigouta, a Tunisian traditional fermented cheese, by standard serial dilutions method and plated on LM17 agar at 30C for 23 days. Isolated colonies were cultivated in LM17 broth and checked for catalase. Catalase-negative strains were selected for bacteriocin screening. Bacteria were removed by centrifugation 10 min at 6000 g and supernatant was checked for antimicrobial activity by agar-well diffusion assay (Tagg et al. 1976). The wells punched on soft agar seeded with 1% of an overnight grown indicator strain were lled with 50 ll of culture supernatant. The plates were incubated overnight at optimal temperature for the indicator strain. At the end of the incubation time, the diameters of the inhibitory zones were measured. For active supernatants, a new antimicrobial test was conducted to eliminate hydrogen peroxide and low pH effects, by adding beef leaver catalase (50 U ml)1) and adjusting the pH value to 60. Only bacteriocin positive strains were checked for Gram-staining, microscopic morphology analysis and carbohydrate fermentation using the API 50 CHL system (bioMerieux, Marcy lEtoile, France). Effect of temperature and protease on antimicrobial activity Bacteriocin containing culture supernatants were submitted to heating at 65C for 30 min and at 100C for 15 min or protease treatment by: trypsin, proteinase K and pronase E at a nal concentration of 1 mg ml)1 for 1 h at 30C. The remaining activity was measured by the agar-well diffusion method towards L. ivanovii BUG496. Untreated preparation of bacteriocin was used as the control. Bacteriocin purication, molecular mass determination and activity titration Bacteriocin purication was perfomed using the three-step procedure developed by Guyonnet et al. (2000). Fractions with bacteriocin activity were concentrated by lyophilization and resuspended in 50% acetonitrile01% formic acid, then analysed by mass spectrometry on a Perkin-Elmer Sciex API 165 mass spectrometer (Boston, MA, USA) equipped with an ion spray source. The sample analysis was carried out by using infusion pump syringe at a ow rate of 5 ll min)1. The instrument scale for the mass-to-charge (m/z) ratio was calibrated with the ions of the ammonium adduct of polypropylene glycol. Scan data were obtained with Tune

12, and mass calculation was peformed with Biomultiview 12 (Sciex, Concord, ON, Canada). The protein concentration was determined by the bicinchoninic acid procedure as described by the supplier with bovine serum albumin as a standard (Sigma). The critical dilution assay was used for bacteriocin titration (Fremaux et al. 1995). A total volume of 50 ll of a serially diluted bacteriocin preparation were deposited into the wells cut in cooled soft agar plates seeded with L. ivanovii BUG496 (1% of overnight cultures, v/v). The plates were examined for zones of inhibition after overnight incubation at 37C. The minimum inhibitory concentration (MIC) was dened as the rst concentration of bacteriocin showing no zone of inhibition around the well. DNA manipulations Plasmid DNA was extracted from LAB by the Anderson and McKay (1983) method. Primers for PCR amplication of nis genes were designed from published sequence (Genbank accession number L16226). The two primers are Prim-NisP5 GGATTTGGTATCTGTTTCGAAG and Prim-NisP3 TCTTTCCCATTAACTTGTACTGTG. The PCR conditions included denaturing at 96C for 30 s followed by annealing at 45C for 30 s, and extension at 72C for 45 s. Reactions (25 cycles) were carried out in a 25-ll volume with a Perkin Elmer GenAmp 2400 PCR system. Other standard techniques were those of Sambrook et al. (1989).

RESULTS Antibacterial activity detection and strains identication Six hundred LAB isolated from rigouta cheese were screened for bacteriocin-like substance production by the agar-well diffusion method. Only eight isolates were found to produce antilisterial activity. Inhibitory spectrum of supernatant for the eight isolated strains is shown in Table 2. Seven strains MMT 01 to 04 and 06 to 08, collectively named group A strains, exhibited similar inhibitory spectrum. They were active against L. ivanovii BUG496, L. monocytogenes EGDe, Ent. faecalis JH2-2, Ent. faecium ENSAIA631, L. lactis subsp. cremoris ATCC 11603, Brochotrix sp. and Serratia sp. but not towards Lact. casei DSM20011, Lact. delbruekii DSM20081, E. coli XL1 Blue, Hafnia sp. and L. lactis subsp. lactis ATCC11454. On the basis of biochemical and morphological characteristics, all the group A strains were identied as L. lactis according to Schleifer and Kilpper-Balz (1987). The seven strains were able to grow on lactose-containing medium and to coagulate milk, indicating that these

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Table 2 Inhibitory spectrum of bacteriocinogenic lactic acid bacteria isolated from rigouta cheese Diameter of inhibition zone (mm) MMT01; 02; 03; 04; 06; 07 and 08 00 155 00 00 140 140 150 110 150 150 00 00 L. lactis ATCC 11454* 0 0 150 0 0 0 0 145 145 165 120 130 150 0 0 0 0 Ped. acidilactici 1521 00 00 00 00 00 210 210 190 00 00 00 00 Leuc. mesenteroides Y105 0 0 0 0 0 0 0 0 180 200 210 200 0 0 0 0 0 0 0 0

Indicators strains L. lactis ATCC11454 L. cremoris ATCC11603 Lact. casei DSM20011 Lact. delbruekii DSM20081 Ent. faecium ENSAIA631 Ent. faecalis JH2-2 L. ivanovii BUG496 L. monocytogenes EGDe Serratia sp. Brochotrix sp. Hafnia sp. E. coli XL1-blue

MMT05 00 00 00 85 105 105 120 110 00 00 00 00

*Producer of nisin A. Producer of pediocin PA-1. Producer of mesentericin Y105 and mesentericin B105.

Absorbency (220 nm)

1400 1200 1000 800 600 400 200 0 5 10 15 20 25 Time (min) 30 35 40 0 235 min 40

Bacteriocins purication and characterization Effect of heat as well as proteolytic enzymes on antilisterial compound of the seven cell-free supernatants from the group A strains was checked. In all cases, antimicrobial activities appeared heat stable but was dissipated by proteinase K and pronase E digestion. Trypsin had no effect on the antilisterial activities. These results demonstrate that the inhibitory activity of the seven strains is related to heat-stable proteinaceous compounds, probably bacteriocins. Moreover, inhibitory spectrum identities suggested that the group A strains produced a similar antagonistic molecule. The active compounds secreted by the seven strains were separately puried to homogeneity by

Fig. 1 Reverse-phase HPLC 220-nm prole of the active fraction obtained from cation exchange chromatography and solid phase extraction on C18 cartridge from culture supernatant of Lactococcus lactis MMT01. Elution was achieved in 45 min at a ow rate of 08 ml min)1 with a 2080% linear gradient of acetonitrile in 01% triuoroacetic acid/water

2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 621628, doi:10.1111/j.1365-2672.2004.02347.x

Acetonitrile (%)

lactococcal strains are able to metabolize lactose and caseinate (Lac+, Prt+ phenotype). The last strain, MMT05, exhibited a different inhibitory prole. It inhibits only ve of the 12 indicator strains (Table 2), i.e. L. ivanovii BUG496,, L. monocytogenes EGDe, Ent. faecalis JH2-2 and Ent. faecium ENSAIA631, Lact. delbruekii DSM20081. MMT05 was initially identied as Lact. paracasei on the basis of carbohydrate fermentation. However, microscopic morphology appeared not consistent with such identication. Indeed bacterial cells appeared as short chains of cocci instead of bacilli. Finally, strain identication was achieved by 16S RNA sequence analysis culaire performed by the Laboratoire dIdentication Mole ries de lInstitut Pasteur de Paris (France). des Bacte Comparison of the rrs gene of the MMT05 strain with data bank sequences strongly suggested that this strain is an Ent. faecalis (data not shown).

a three chromatographic steps procedure (Guyonnet et al. 2000). The elution prole obtained at the ultimate reversephase HPLC step appeared identical for the seven strains and is presented in Fig. 1. The fraction corresponding to the 245-min peak showed antimicrobial activity and was subjected to mass spectrometry analysis. The molecular mass of this active peptide is 333119 058 Da (Fig. 2a),

2200 2000 1800 1600

245 min

80

ANTILISTERIAL ACTIVITY OF LAB ISOLATED FROM RIGOUTA

625

(a)

[M + 3H+]3+
11113

10

15e6 14e6 13e6 12e6 [M + 4H+]4+ 11e6 8339 10e6 90e5 80e5 70e5 60e5 50e5 40e5 6849 30e5 20e5 10e5

bp 600 500 Molecular mass M = 333119 058 Da

Intensity (cps)

[M + 2H+]2+ 16665

22219 19560 2523326659 28675

Fig. 3 Agarose gel electrophoresis of PCR products obtained with Prim-NisP5 and Prim-NisP3 primers. The DNA used as template were prepared from Lactococcus lactis: lane 1: ATCC 11454 (nisin A producing strain); lane 2: MMT08; lane 3: MMT07; lane 4: control sample (no DNA); lane 5: DNA marker; lane 6: MMT06; lane 7: MMT04; lane 8: MMT03; lane 9: MMT02; lane 10: MMT01

600 900 1200 1500 1800 2100 2400 2700 m/z (b)
[M + 3H+]3+ 11165

36e6 34e6 32e6 30e6 28e6 + 4+ 26e6 [M + 4H ] 8379 24e6 22e6 20e6 Molecular mass M = 33469 040 Da 18e6 16e6 14e6 12e6 [M + 2H+]2+ 10e6 16744 80e5 60e5 40e5 20e5 22327 600 900 1200 1500 1800 2100 2400 2700 m/z

Fig. 2 Mass spectrometry analysis of puried bacteriocins from Lactococcus lactis MMT01 with multiple charged molecular ions [M + nH+]n. (a) Electrospray ionization mass spectrum of fraction 245 min. (b) Electrospray ionization mass spectrum of fraction 235 min

that corresponds to nisin Z. In all the seven samples, a 235min peak, not fully resolved from the 245-min peak, was detected (Fig. 1). Mass spectrometry analysis of this fraction gave a molecular mass of 334699 040 Da (Fig. 2b). Database screening revealed that none of the described bacteriocins produced by LAB correspond to this molecular mass. MIC of nisin Z, was estimated, by the well-diffusion method against L. ivanovii BUG496, to 420 ng ml)1. In order to conrm the nisin Z production by the group A strains, PCR analysis was conducted using whole DNA

preparation as matrix, and Prim-Nis5 and Prim-Nis3 primers designed from published sequence of the structural nis genes (Genbank accession number L16226). As predicted, these two primers lead to amplify a 598-bp fragment from the DNA of L. lactis subsp. lactis ATTCC 11454, a nisin A producer (Fig, 3, lane 1). Similar fragment is amplied from the DNA of the group A strains (Fig. 3, lanes 23 and 610) but not from DNA of Ent. faecalis MMT05 nor Lactobacillus sakei 2525 (Guyonnet et al. 2000), that secretes sakacin P a class IIa bacteriocin (Tichaczek et al. 1994; data not shown). Furthermore, each strain of this antilisterial seven-strain group appeared to be insensitive to the culture supernatant of other group A strains or L. lactis subsp. lactis ATCC11454, in agar-well diffusion assay (data not shown). Finally, the group A strains produced nisin Z (Nis+) and were resistant to nisin (NisR). Plasmid proles of the seven group A strains were compared in order to determine if these lactococcal strains correspond to several isolates. From the seven strains only three different proles were observed (Fig. 4), demonstrating the existence of, at least, three genetically different strains. The three groups were composed of the following strains (i) MMMT01, 02, 03 and 07; (ii) MMT 04 and 08; and (iii) MMT06. Contrarily to nisin Z, the antibacterial compound produced by MMT05 appeared heat sensitive and was inactivated after incubation with each of the three enzymes, proteinase K, pronase E and trypsin. This proteinaceous compound was puried using the Guyonnet et al. (2000) method. Antilisterial activity was detected in the fraction corresponding to a 24-min peak from reverse-phase elution chromatography. The fraction was then subjected to molecular mass determination. Mass spectrometry analysis gave a molecular mass of 10 20133 085 Da (Fig. 5). Then, MIC of this compound produced by MMT05 was determined against L. ivanovii BUG496 and was equal to 677 ng ml)1.

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Intensity (cps)

626 T . G H R A I R I ET AL.

kbp

8 7 6 5 4 3 2

Fig. 4 Plamid proles of Lactococcus lactis strains isolated from Tunisian fermented cheese rigouta. Lane 1: MMT06; lane 2: MMT07; lane 3: MMT04

[M + 13H+]13+

8511 [M + 12H+]12+ 7858

14e6 13e6 12e6 11e6 10e6 90e5 80e5 70e5 60e5 50e5 40e5 30e5 20e5 10e5

[M + 14H+]14+

Intensity (cps)

7298

9283 [M + 11H+]11+

Molecular mass M = 1020133 085 Da

10212 [M + 10H+]10+ 11348 [M + 9H+]9+ 14584

600

800

1000

1200 m/z

1400

1600

1800

Fig. 5 Mass spectrometry analysis of puried bacteriocin from Enterococcus faecalis MMT05 showing multiple ions [M + nH+]n at different m/z values

DISCUSSION On the basis of biochemical and morphological characteristics, all the group A strains were identied to L. lactis, as all the nisin producer strains from natural isolates, in our knowledge. However, these identications need to be conrmed by molecular methods, e.g. RAPD or molecular analysis of specic genes (Corroler et al. 1999). All these seven strains are characterized by the production of nisin Z (molecular mass 333119 058 Da). Insensitivity to trypsin degradation of this peptide is most probably because of the presence of post-

translationally modied residues (McAuliffe et al. 2001). A less abundant peptide was detected with a molecular mass of 334699 040 Da. The measured molecular mass for the two peptides differed by only 16 Da. Moreover, nisin contains methionine residues which are well known to be susceptible for oxidation Taken together, these data strongly suggest that the 3347-Da peptide corresponds most probably to a monooxidized form of nisin Z. The purication strategy used herein was originally designed for class IIa bacteriocins (Guyonnet et al. 2000). However, in this study this rapid method was also efcient for lantibiotics purication. Nisin and sucrose genes were shown to be linked into a conjugative transposon in L. lactis (Horn et al. 1991; Rauch and de Vos 1992). Such a link was demonstrated for natural nisin producers in conjugal transfer mating (Olasupo et al. 1999). The group A strains were able to ferment sucrose as shown by their ability to grown on SM17 medium. This data suggests the possibility that these strains possess such a nisinsucrose transposon. Genetic transfer of the putative nisinsucrose transposon could be responsible for the presence of different nisin Z-producing strains in the same environment. The entire group A strains is able to grow in milk and is composed, at least, by three different strains. Taken together, presence of different strains in this group and their phenotype, i.e. Nis+ NisR Lac+ Prt+, indicates that these strains could constitute promising candidates to design starter blends for use in cheese manufacturing. Additional experiments to determine their technological properties are planned. MMT05 strain was identied at the molecular level as Ent. faecalis. The antilisterial thermo-labile protein produced by this strain was named enterocin MMT05. Measured molecular mass of this compound (10 201 Da) strongly suggests that it is a new member of class III bacteriocins, the rst one described produced by wild LAB isolated from traditional Tunisian foods. Puried nisin Z and enterocin MMT05 were tested toward the same indicator, L. ivanovii BUG496, in order to compare their specic activities. Specic activity of nisin Z (420 ng ml)1) appeared higher than enterocin MMT05 one (677 ng ml)1). MICs of some class IIa bacteriocins were determined against the same indicator strain (Guyonnet et al. 2000; Ferchichi et al. 2001). It appears, among the tested bacteriocins, that enterocin MMT05 displayed the higher MIC value. This result depends on a lower specic activity for enterocin MMT05, or/and to a weak diffusion capability around the wells punched in agar related to the higher molecular mass of this molecule (10 201 Da) compared with that of antilisterial peptides. In cheese manufacturing, high level of contaminating enterococci usually results from poor hygienic practices. These bacteria lead to deterioration of organoleptic proper-

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ties in some kind of cheeses but were demonstrated to play a major role in ripening and aroma development in other cases (Giraffa 2002). This benecial role has led to design-specic starter cultures including enterococcal strains, from species Ent. faecalis and Ent. faecium (Franz et al. 1999). As Enterococcus strains can predominate during ripening of cheeses, bacteriocinogenic enterococci may be used as antilisterial agents in the dairy industry (Foulquie Moreno et al. 2003) and could produce bacteriocin at sufcient level to inhibit Listeria (Franz et al. 1999). Nevertheless, enterococci could exert detrimental role in food, selection of Ent. faecalis MMT05 strain in cheese making will demand additional studies to demonstrate its safety use. AKNOWLEDGEMENTS Mixte Taouk Ghrairi is supported by grant from the Comite ration Universitaire Franco-Tunisen (CMCU). We de Coope thanks Daniel Guyonnet for mass spectral analysis. REFERENCES
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2004 The Society for Applied Microbiology, Journal of Applied Microbiology, 97, 621628, doi:10.1111/j.1365-2672.2004.02347.x

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