Sardinian goat’s milk as source of bacteriocinogenic potential protective cultures

Food Control 25 (2012) 309e320

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Sardinian goat’s milk as source of bacteriocinogenic potential protective cultures Stefano Schirru a, Svetoslav Dimitrov Todorov b, *, Lorenzo Favaro c, Nicoletta Pasqualina Mangia d, Marina Basaglia c, Sergio Casella c, Roberta Comunian a, Bernadette Dora Gombossy de Melo Franco b, Pietrino Deiana d a

AGRIS Sardegna e Dipartimento per la Ricerca nelle Produzioni Animali, Località Bonassai, Olmedo (SS), Italy Universidade de São Paulo, Faculdade de Ciências Farmacêuticas, Departamento de Alimentos e Nutrição Experimental, Laboratório de Microbiologia de Alimentos, São Paulo (SP), Brazil c Università degli Studi di Padova, Facoltà di Agraria, Dipartimento di Biotecnologie Agrarie, Legnaro (PD), Italy d Università degli Studi di Sassari, Facoltà di Agraria, Dipartimento di Scienze Ambientali Agrarie e Biotecnologie Agro-Alimentari (DiSAABA), Sassari (SS), Italy b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 June 2011 Received in revised form 21 October 2011 Accepted 30 October 2011

Goat breeding in Sardinia constitutes an important source of income for farming and shepherding activities. In this study 170 LAB strains were isolated from Sardinian goat’s milk and tested for bacteriocins production against several food-borne pathogenic microorganisms. Four isolates (SD1, SD2, SD3 and SD4) were selected for their effective inhibition on Listeria monocytogenes. The strains were classified as members of Enterococcus genus, according to their biochemical and physiological characteristics, and then genetically identified as Enterococcus faecium. In MRS broth at 37
C, bacteriocins SD1 and SD2 were produced at much higher levels (51200 AU/ml) compared to bacteriocin SD3 (3200 AU/ml) and bacteriocin SD4 (800 AU/ml). Their peptides were inactivated by proteolytic enzymes, but not when treated with a-amylase, catalase and lipase. The four bacteriocins remained stable at pH from 2.0 to 12.0, after exposure to 100
C for 120 min and were not affected by the presence of surfactants and salts (NLaourylsarcosine, NaCl, SDS, Triton X-100, Tween 20, Tween 80 and urea). Their molecular size was determined to be approximately 5 kDa by tricine-SDS-PAGE. Since the strains exhibited a strong antimicrobial activity against 21 L. monocytogenes strains and 6 Salmonella spp. isolates, they should be considered as potential bio-preservatives cultures for fermented food productions. Moreover, due to their technological features, the four strains could be taken in account for using as adjunct NSLAB (non-starter lactic acid bacteria) rather than as starter culture. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Enterococcus faecium Bacteriocin Goat’s milk

1. Introduction Due to the widely appreciated organoleptic characteristics, the production of goat’s milk, intended for direct consumption and cheese processing, has attracted growing interest over recent years. Sardinia, the second largest island in the Mediterranean Sea, is one of the major goat’s milk-producing regions in Italy. Sardinia’s goat population stands at about 330,000, representing 25% of the Italian goats and on average, the Sardinian dairy processing industry manufactures more than 50% of the Italian processed goat’s milk (ISTAT, 2006). Lactic acid bacteria (LAB) occur naturally as indigenous microbiota in raw milk and several strains associated with food systems produce bacteriocins able to inhibit spoilage and pathogenic

* Corresponding author. Tel.: þ55 11 3091 2191; fax: þ55 11 3815 4410. E-mail address: [email protected] (S.D. Todorov). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.10.060

microorganisms (Jack, Tagg, & Ray, 1995). Among LAB, Enterococci are natural inhabitants of the gastrointestinal tract of humans and other animals, and are also present in vegetables, plant material and foods (Giraffa, 2002). They are commonly found in high levels in a variety of cheeses produced in Italy, Portugal, Spain and Greece from raw or pasteurized goats’, ewes’, water buffalo or cows’ milk and are thought to positively influence the flavor of cheese (Foulquié Moreno, Sarantinopoulos, Tsakalidou, & De Vuyst, 2006; Franz, Holzapfel, & Stiles, 1999; Franz, Stiles, Schleifer, & Holzapfel, 2003; Giraffa, 2002; Macedo, Malcata, & Hogg, 1995; Mangia, Murgia, Garau, Sanna, & Deiana, 2008; Suzzi et al., 2000). Moreover, the enterococci may produce antibacterial peptides (bacteriocins), generally called enterocins. Bacteriocins of LAB are defined as ribosomally synthesized proteins or protein complexes usually antagonistic to genetically closely related organisms (De Vuyst & Vandamme, 1994, p. 539). Bacteriocins are generally low molecular weight proteins that gain entry into target cells by binding to cell surface receptors, and can act by different

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bactericidal mechanisms (De Vuyst & Vandamme, 1994, p. 539). In recent papers, specific environmental conditions, including those found in food, have been studied to determine their effect on the production of bacteriocins (Leroy & De Vuyst, 2003; Motta & Brandelli, 2003). Bacteriocin production dramatically changes upon altering of environmental conditions and optimum production may require a precise combination of specific parameters (Leal-Sánchez, Jiménez-Díaz, Maldonado-Barragán, GarridoFernández, & Ruiz-Barba, 2002). Interest in bacteriocins produced by some LAB has been stimulated by the fact that they are active against Gram-positive foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, and vegetative cells and spores of Clostridium botulinum (Chen & Hoover, 2003; Deegan, Cotter, Hill, & Ross, 2006). However, despite the fact that enterococci have a long history of safe use, they are not generally recognized as safe (GRAS) organisms, because they have been reported as emerging pathogens associated with nosocomial infections and superinfections such as endocarditis, bacteremia, and urinary tract and other infections (Franz & Holzapfel, 2004; Ogier & Serror, 2008). The objective of this study was to isolate bacteriocin-producing LAB strains with antibacterial activity that could be used as starter or adjunct protective cultures in cheeses production. For the present purpose, a total of 170 enterococci isolated from Sardinian goat’s milk were tested for their inhibitory effects on some pathogenic microorganisms. Furthermore, some aspects of mode of action of Enterocin SD1, Enterocin SD2, Enterocin SD3 and Enterocin SD4 produced by four Enterococcus faecium strains, and several technological features of these strains were studied. 2. Materials and methods 2.1. Bacteria isolation and growth conditions Samples of goat’s milk were serially diluted in sterile physiological water (0.85% NaCl) and plated in duplicate onto Azide dextrose Agar (Oxoid, Milan, Italy) and MRS agar (Oxoid). The plates were incubated at 37
C for 72 h in anaerobic jars using AnaeroGenÔ system (Oxoid). One hundred and seventy isolates were randomly picked up from plates, purified by streaking twice on fresh MRS (De Man, Rogosa, & Sharp, 1960) plates (Oxoid). The isolates were cultured in MRS broth medium (Oxoid) at 37
C and stored at 80
C in spent MRS broth, supplemented with 15% (v/v) glycerol. MRS broth was used in all following experiments. 2.2. Bacteriocin-producing isolates selection All isolates were tested for the production of bacteriocins by agar spot-test and well-diffusion-method against several food-borne pathogenic microorganisms, namely Escherichia coli DSMZ 30083, L. monocytogenes IZPSS, Salmonella choleraesuis DSMZ 13772, S. aureus DSMZ 20231 (Harris, Daeschel, Stiles, & Klaenhammer, 1989). 2.3. Isolates identification and strain typing Isolates SD1, SD2, SD3 and SD4, selected in this study on the base of their ability to produce antibacterial substance/s, were classified on their physiological and biochemical characteristics as described by Bridge and Sneath (1983) and Stiles and Holzapfel (1997) and genetically identified by 16S rDNA sequencing. Genomic DNA was extracted as follows: a small colony of each strain, grown for 24 h on Nutrient Agar (NA) plates, were picked up with a sterile toothpick and resuspended in 50 ml of lysis solution (0.05 M NaOH, 0.25%

SDS). After vortexing for 2 min, the suspension was heated at 95
C (15 min) and then centrifuged (10,000 g, 15 min). PCR amplification was carried out using bacterial universal primers and conditions described by Hongoh, Yuzawa, Ohkuma, and Kudo (2003). Amplification products were checked by agarose gel electrophoresis and then subjected to sequencing. Species identification was done after BLASTN alignment (http://www.ncbi.nlm.nih. gov/BLAST) of the obtained sequences with those present in the GenBank public database. A minimum sequence similarity level of 98% was considered for taxonomic attribution. The species identification was confirmed by species-specific PCR using FM1/FM2 primers pair as described by Jackson, Fedorka-Cray, and Barrett (2004). Strain typing was performed by pulsed-field gel electrophoresis (PFGE) as described by Mannu and Paba (2002). In addition, scanning electron microscopy (SEM) was performed on strains with a Zeiss DSM962 microscopy (Bozzola, 2007). 2.4. Bacteriocin bioassay Bacteriocin screening was performed by using the agar-spottest method. Correction of the cell-free supernatant to pH 6.0 with 1 M NaOH prevented the inhibitory effect of lactic acid. Antimicrobial activity was expressed as arbitrary units (AU/ml), calculated as ab  100, where “a” represents the dilution factor and “b” the last dilution that produces an inhibition zone of at least 2 mm in diameter. Activity was expressed per ml by multiplication with a proper conversion factor. One AU is defined as the reciprocal of the highest dilution showing a clear zone of growth inhibition. L. monocytogens 211 (collection of Department of Food Science and Experimental Nutrition, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil) was used as indicator strain. Cell-free supernatant containing bacteriocins SD1, SD2, SD3 and SD4 was incubated at 26
C for 72 h and at regular intervals bacteriocin activity was determined as described before. In addition the bacteriocins activity was determined against a number of microorganisms, including different LAB strains, L. monocytogenes from different serological groups and Gramnegative bacteria. The list and growth conditions of test strains are specified in Table 1. 2.5. Growth dynamics of strains, bacteriocins production and changes of pH An 18-h-old culture of strains SD1, SD2, SD3 and SD4 were inoculated (2%, v/v) into the following media: MRS broth, skimmed milk (Oxoid) (0.2 and 10%, w/v) and cheese whey (Batavo, Sao Paulo, Brazil) (0.2 and 10%, w/v), respectively and incubated at 26, 30 and 37
C, respectively, without agitation, for 24 h. In addition, dynamics of bacteriocins SD1, SD2, SD3 and SD4 production was studied in MRS broth at 26
C during 24 h. Samples were taken every hour and examined for bacterial growth (OD600nm), pH changes in culture, and antimicrobial activity against L. monocytogenes 211. The agar-spot test method was used and the activity expressed as AU/ml as described previously. All experiments were done in triplicate. 2.6. Adsorption to producer cells The ability of the bacteriocins to adsorb to the producer cells was studied as described by Yang, Johnson, and Ray (1992). The cell-free supernatant was neutralized to pH 7.0 with sterile 1 N NaOH and tested for activity using the agar spot-test method.

S. Schirru et al. / Food Control 25 (2012) 309e320 Table 1 Spectrum of antibacterial activity of bacteriocins SD1, SD2, SD3 and SD4 produced by Enterococcus faecium strains. Test microorganisms

L. monocytogenes 1/2a, 103 L. monocytogenes 1/2a, 104 L. monocytogenes 1/2a, 409 L. monocytogenes 1/2a, 506 L. monocytogenes 1/2a, 709 L. monocytogenes 1/2b, 426 L. monocytogenes 1/2b, 603 L. monocytogenes 1/2b, 607 L. monocytogenes 1/2c, 408 L. monocytogenes 1/2c, 422 L. monocytogenes 1/2c, 637 L. monocytogenes 1/2c, 711 L. monocytogenes 1/2c, 712 L. monocytogenes 4b, 101 L. monocytogenes 4b, 211 L. monocytogenes 4b, 302 L. monocytogenes 4b, 620 L. monocytogenes 4b, 703 L. monocytogenes 4b, 724 L. monocytogenes ScottA L. monocytogenes 38 L. monocytogenes 39 L. welshimeri 12 L. seeligeri 13 Bacillus cereus ATCC 11778 Bacillus mycoides 14 E. faecium SD1 E. faecium SD2 E. faecium SD3 E. faecium SD4 E. faecium ATCC 19443 Enterobacter aerogenes ATCC 13048 Escherichia coli ATCC 8739 L. sakei ATCC 15521 Salmonella Montevideo 28 Salmonella Agona 29 S. enterica subsp. enteric 31 Salmonella Brandenburg 32 Salmonella Brandenburg 33 Salmonella Madelia 34 S. enteritidis ATCC 13076 Salmonella Anatum 30 Salmonella Braenderup H9812 Salmonella Heidelberg 27 Staphylococcus aureus ATCC 25923 S. aureus ATCC 29213 S. aureus ATCC 6538

Growth medium

Growth temp. (
C)

Bacteriocins produced by E. faecium (presented in mm inhibition zones) SD1

SD2

SD3

SD4

BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI MRS MRS MRS MRS MRS BHI

37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 37 30 30 30 30 30 37

20 21 21 15 22 20 21 22 20 18 17 23 20 18 17 15 18 16 20 0 0 0 0 0 0 0 0 0 17 0 8 0

15 22 18 15 20 18 18 23 18 15 20 18 20 19 12 12 15 18 19 0 0 0 0 0 0 0 0 0 16 0 0 0

11 8 17 12 12 12 12 17 12 12 15 10 10 15 10 10 16 10 10 0 0 0 0 0 0 0 17 10 0 0 0 0

0 0 0 0 0 0 0 5 0 0 5 0 0 0 4 0 5 0 0 0 0 0 0 0 0 0 10 0 9 0 0 0

BHI MRS BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI BHI

37 30 37 37 37 37 37 37 37 37 37 37 37

0 9 12 11 15 10 13 8 0 0 0 0 0

0 0 14 10 16 9 14 9 0 0 0 0 0

0 0 6 0 9 7 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0

BHI BHI

37 37

0 0

0 0

0 0

0 0

BHI: Brain Heart Infusion agar (Oxoid), MRS: De Man, Rogosa, Sharpe agar (Oxoid).

2.7. Determination of approximate molecular weight of bacteriocins by SDS-PAGE The selected strains were grown in MRS broth for 24 h at 37
C. The cells were harvested by centrifugation (10,000 g, 15 min, 4
C) and the bacteriocin precipitated from the cell-free supernatants with 50% ammonium sulfate. The precipitate was resuspended in one-tenth volume 25 mM ammonium acetate (pH 6.5), desalted against distilled water by using a 1000 Da cut-off dialysis membrane (Spectrum Inc., CA, USA) and separated by tricine-SDSPAGE, as described by Schägger and von Jagow (1987). A molecular weight marker with sizes ranging from 10 kDa to 75 kDa was used. The gels were fixed and one half overlaid with L. monocytogens 211

311

(106 CFU/ml), embedded in BHI agar (Oxoid), to determine the position of the active bacteriocin, and the second half of the gel stained with Comassie Blue R250, as described by Todorov et al. (2010). 2.8. Effect of enzymes, chemicals, pH and temperature on the bacteriocins activity Cell-free supernatants of SD1, SD2, SD3 and SD4 strains, obtained by centrifugation (10,000 g, 15 min, 4
C) were adjusted to pH 6.0 with 1 N NaOH. Aliquots of 2 ml were incubated for 2 h in the presence of 1.0 mg/ml (final concentration) of trypsin, pronase, proteinase K, pepsin, papain, catalase, lipase and a-amylase (all from Sigma) and then tested for antimicrobial activity using the agar-spot test method against L. monocytogenes 211. In another experiment, the effect of laourylsarcosine, NaCl, SDS, Triton X-100, Tween 20, Tween 80 and urea on bacteriocins activity in cell-free supernatants was determined as described by Todorov et al. (2010). The effect of pH on the bacteriocins was determined by adjusting the cell-free supernatant between pH 2.0 and 12.0 with sterile 1 N HCl or 1 N NaOH. After 2 h of incubation at 30
C, the samples were readjusted to pH 6.5 and the activity determined as described before. The effect of temperature on the bacteriocins was tested by heating the cell-free supernatants to 60, 70, 80, 90 and 100
C for 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 120 min. In addition, residual bacteriocin activity was tested also after 15 min at 121
C as described elsewhere (Todorov et al., 2010). In all the above mentioned studies, uninoculated MRS broth was exposed to the same conditions (digestive enzymes, chemicals, pH and temperature) as well as the cell-free supernatants and used as a control against L. monocytogenes 211. All chemicals and enzymes were supplied by Sigma. 2.9. Determination of the reduction of target microorganisms in the presence of bacteriocins Twenty milliliters cell-free supernatant containing bacteriocins SD1, SD2, SD3 and SD4 (pH 6.0) was filter-sterilized (0.20 mm, MinisartÒ, Sartorius) and added to 100 ml 3-h-old cultures (OD600nm ¼ 0.1e0.2) of L. monocytogenes 211. Incubation was on BHI broth at 37
C. Optical density readings were recorded at 600 nm, hourly for 12 h. The experiment was repeated with stationaryphase cells. In a separate experiment, early stationary phase (18-h-old) cultures of L. monocytogenes 211 (sensitive strain) and L. monocytogenes ScottA (resistant strain) were harvested (5000 g, 5 min, 4
C), respectively, washed twice with sterile saline water and resuspended in 10 ml. Equal volumes of the cell suspensions and filter-sterilized bacteriocins SD1, SD2, SD3 and SD4 containing cell-free supernatant were mixed. Viable cell numbers were determined before and after incubation for 1 h at 37
C by plating onto BHI supplemented with 1.5% agar. Cell suspensions of L. monocytogenes 211 and L. monocytogenes ScottA without added bacteriocins served as controls. 2.10. Adsorption of bacteriocins to target cells Adsorption of bacteriocins SD1, SD2, SD3 and SD4, to target cells was performed according to the method described by Yildirim, Avs¸ar, and Yildirim (2002), using the target strains L. monocytogenes 211, L. monocytogenes 637, L. monocytogenes 620, Lactobacillus sakei ATCC 15521 and E. faecium ATCC 19443. The activity of unbound bacteriocins in the supernatant was

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determined as described before. All experiments were done in duplicate. The percentage of adsorption of bacteriocins to target cells was calculated according to the following formula:

 % adsorption ¼ 100 

 AU=ml1  100 AU=ml0

AU/ml1 refers to the bacteriocin activity after treatment; AU/ml0 refers to the original (before treatment) activity. 2.11. Effect of pH, temperature, inorganic salts and organic compounds on the adsorption of bacteriocins to L. monocytogenes 211 cells The bacteriocins were added to L. monocytogenes 211, as described previously, and incubated for 1 h at 4, 25, 30 and 37
C, respectively (pH 6.0), and at 30
C at pH 4.0, 6.0, 8.0 and 10.0. Cells were harvested (8000 g, 15 min, 25
C) and the pH of the cell-free supernatant adjusted to 6.0 with sterile 1 M NaOH. Bacteriocins activity in the supernatants was determined as described before. The experiments were done in duplicate. L. monocytogenes 211 cells were also treated with 1% (m/v) Tween 20, Tween 80, NaCl, SDS, Na2HPO4 and NaH2PO4. The pH of all samples were adjusted to 6.5 with 1 M NaOH or 1 M HCl. Bacteriocins were added to the treated cells, as described before, and incubated for 1 h at 30
C. The cells were harvested (8000 g, 15 min, 25
C) and the activity of bacteriocins in the cell-free supernatants determined as described before. The experiments were done in duplicate. 2.12. Growth at different pH values and NaCl concentrations The four strains were grown in MRS broth adjusted to pH 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively. The pH was adjusted with 1 M NaOH or 1 M lactic acid (Sigma) before autoclaving and readjusted after autoclaving if the pH changed by more than 0.2 units. Tolerance to NaCl was tested by growing the cells in MRS broth supplemented with increasing concentrations of sodium chloride (1-2-3-4-5 and 6%). All tests were conducted in STERELINÔ microtiter plates incubated in TECAN SPECTRAFLUOR (Milan, Italy) equipment. Each well was filled with 180 ml of the MRS medium and inoculated with 20 ml overnight pre-culture (OD600nm ¼ 0.02). Optical density readings (at 600 nm) were recorded every hour for up to 50 h. The growth experiments were conducted in triplicate. Cultures grown in MRS broth served as control. 2.13. Acidifying, lipolytic and proteolytic activity of the selected strains The four strains were initially grown in BHI broth and then in 100 ml sterile reconstituted skimmed milk (10% w/v) inoculated with 1% of an overnight activated culture, and pH changes were determined (Hanna Instruments 302 pH METER, Woonsocket, RI, USA) during incubation at 37
C after 3, 6, 9 and 24 h. These values were also compared to the values gained in MRS and BHI broths after 24 h of incubation. Lipolytic activity was tested using Tributyrin agar (Oxoid) as described by (Charoenchai, Fleet, Henschke, & Todd, 1997). Proteolytic activity was determined using the o-phthadialdehyde spectrophotometric assay (OPA) as described by Church, Swaisgood, Porter, and Catignani (1983). All experiments were conducted in triplicate.

2.14. Susceptibility of strains to antibiotics Susceptibility of strains SD1, SD2, SD3 and SD4 to several antibiotics (listed in Table 4) was tested by the disk diffusion test, using disks from CEFAR (São Paulo, Brazil). Each strain was inoculated into 10 ml MRS broth and incubated at 37
C for 18 h and mixed into MRS soft agar (1.0%, w/v), in order to achieve a population of 106 CFU/ml. After solidification of the agar, each antibiotic disk was spotted onto the surface of the plates, and incubated at 37
C for 24 h. The plates were examined for the presence of inhibition zones around the antibiotics disks. The inhibitory effect of the antibiotics was expressed in millimeters of the inhibition zones. 3. Results and discussion 3.1. Isolation and identification of LAB from goat’s milk Overall, 170 LAB were isolated from Sardinian goat’s milk and successfully identified as Enterococcus by physiological and biochemical tests. Four isolates (SD1, SD2, SD3 and SD4) were selected for their effective inhibition on L. monocytogenes. Their morphology was visualized by SEM (Fig. 1). They were genetically identified as E. faecium by 16S rDNA sequencing and speciesspecific PCR. The four isolates resulted to be different strains by PFGE analysis (Fig. 2). 3.2. Characterization of bacteriocins Bacteriocins SD1, SD2, SD3 and SD4 showed a large spectrum of activity, inhibiting the growth of many food spoilage bacteria and food-borne pathogens (Table 1). The cell-free supernatant of E. faecium SD1 inhibited the growth of L. monocytogenes 1/2a, 1/2b, 1/2c and 4b, E. faecium SD3, L. sakei, Salmonella enterica subsp. enteric, Salmonella Montevideo, Salmonella Agona, Salmonella Brandenburg and Salmonella Madelia. Similar inhibition profiles were observed for cell-free supernatant from E. faecium SD2 and E. faecium SD3 with the exception that both strains did not show antimicrobial activity against E. faecium ATCC 19443 and L. sakei ATCC 15521. A narrow spectrum of activity was recorded for the cell-free supernatant of E. faecium SD4 as inhibiting the growth only of L. monocytogenes 1/2b, 1/2c, 4b and E. faecium SD1 and SD3 (Table 1). In order to apply this bacteriocin producer strains (SD1, SD2, SD3 and SD4) in co-culture with commercial starters will be essential to confirm not antagonistic properties between them. Taking in consideration that bacteriocin activity is very strain specific, activity cross check will need to be investigated for any specific case. The inhibition of L. monocytogenes indicates that the produced bacteriocins may belong to the class IIA (Anti-Listerial bacteriocins) with the presence of the conservative N-terminal region YGNGV in their structure (De Vuyst & Vandamme, 1994, p. 539; Todorov, 2009). The activity of the selected bacteriocin-producing strains against Gram-negative bacteria should be considered unusual since it has been previously reported for only few bacteriocins, e.g. enterocins produced by Enterococcus faecalis strains (Ananou, Gálvez, Martýnez-Bueno, Maqueda, & Valdivia, 2005; Sparo et al., 2006), enterocin 012 produced by Enterococcus gallinarum (Jennes, Dicks, & Verwoerd, 2002), and bacteriocins produced by Enterococcus mundtii (De Kwaadsteniet, Todorov, Knoetze, & Dicks, 2005; Knoetze, Todorov, & Dicks, 2008; Todorov, Wachsman, Knoetze, Meincken, & Dicks, 2005). In the work of Todorov et al. (2005), activity of bacteriocin produced by E. mundtii ST4V against Gram-negative bacteria (Pseudomonas aeruginosa and Klebsiella pneumoniae) and viruses [herpes simplex viruses HSV-1 (strain F) and HSV-2 (strain G), a polio virus (PV3, strain Sabin)

S. Schirru et al. / Food Control 25 (2012) 309e320

313

Fig. 1. Morphology of E. faecium SD1 (A), E. faecium SD2 (B), E. faecium SD3 (C) and E. faecium SD4 (D) visualized by Scanning Electron Microscopy (SEM).

and a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of MV)] has been reported. Such as activity is unusual, however, De Kwaadsteniet et al. (2005) as well reported that bacteriocin ST15 produced by E. mundtii ST15 (most probably identical to E. mundtii ST4V) was active against Acinetobacter baumanii, K. pneumoniae and P. aeruginosa (De Kwaadsteniet et al., 2005). Authors reported to the same spectrum of activity for bacteriocin ST15 fractions collected from the gel filtration chromatography. The mode of activity could possibly be the destabilization of the plasma membrane, as observed for many other bacteriocins (De Vuyst & Vandamme, 1994, p. 539). Furthermore, as indicated in Table 1, the strong activity of the bacteriocins SD1, SD2, SD3 against L. monocytogenes and Salmonella spp. may have a positive effect on the food safety if the selected strains would be applied for cheese production.

Fig. 2. PFGE profile of E. faecium SD1, E. faecium SD2, E. faecium SD3 and E. faecium SD4. Marker used is a mix of the following New England Biolabs ladders: Lambda Ladder PFG Marker (N0340S) þ Low Range Marker (N0350S).

3.3. Bacteriocin production Once cultured for 24 h in MRS broth at 26
C, 30
C or 37
C, each strain showed similar growth and bacteriocin production (data not shown). This is in accordance with the data previously reported for mundticin ST4SA (Todorov & Dicks, 2009b). Based on these results, all further experiments were conducted at 37
C. Growth of E. faecium strains in 0.2 and 10% skimmed milk or 0.2 and 10% cheese whey was very similar to the growth observed on MRS (data not shown). When grown in MRS broth at 26
C, the cell density of the 4 strains increased from OD600 0.04e0.05 to approximately 2.7e3.0 during 24 h (Fig. 3AeD). During the growth, the pH of the medium decreased from 6.5 to about 4.3. Important differences in the amounts of produced bacteriocin activities, recorded against L. monocytogenes 211, were found between the strains. Low levels of bacteriocins SD1, SD2 activity (800 AU/ml) were detected after 3 h of growth (Fig. 3A and B). For strain SD3 activity level of 400 AU/ml was recorded at 6 h (Fig. 3C) and the strain SD4 produced only 200 AU/ml after 15 h (Fig. 3D). The maximum SD1 and SD2 bacteriocin production (51,200 AU/ml) was obtained after 21 h incubation (Fig. 3A and B) while strain SD3 produced the highest bacteriocin activity (3200 AU/ml) at 15 h (Fig. 3C). Bacteriocin SD4 showed its maximum activity (800 AU/ml) after 21 h (Fig. 3D). With the exception of the latter bacteriocin, a decrease in activity was observed once grown the strains for period longer than 21 h (Fig. 3AeC). For some other bacteriocins similar results were reported, suggesting that the selected bacteriocins may be primary metabolites (Todorov, 2009; Todorov & Dicks, 2004). For instance, optimal production of bacteriocin AMA-K (25,600 AU/ml) from Lactobacillus plantarum AMA-K was recorded after 29 h growth in MRS broth and a significant decrease of activity was later on detected (Todorov, 2008). Several studies have shown that bacteriocin production is dependent on the cell biomass. Todorov and Dicks (2009b) reported

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Fig. 3. Production of bacteriocin SD1 (A), SD2 (B), SD3 (C) and SD4 (D) in MRS broth (pH 6.5 at 26
C). Antimicrobial activity against L. monocytogenes 211 is presented as AU/ml (bars). Modifications in OD (:) and pH (A) are indicated.

that optimal levels of mundticin ST4SA were obtained in growth media that supported high biomass production, such as MRS. The decrease in activity of bacteriocins SD1, SD2, SD3 might be explained considering their degradation by extracellular proteolytic enzymes. Similar decrease has been observed for the bacteriocins secreted by E. faecium ST5Ha (Todorov et al., 2010), E. mundtii ST4SA (Todorov & Dicks, 2009b).

3.4. Adsorption of bacteriocins to the producer cells The ability of the bacteriocin to adsorb to the producer cells is an important feature that may have application in purification processes. Treatment of the cultures of E. faecium strains with 100 mM NaCl (pH 2.0) for 1 h caused adsorption of the bacteriocin to the cells at 200 AU/ml. Different results are reported in literature

S. Schirru et al. / Food Control 25 (2012) 309e320

for L. plantarum ST16Pa (400 AU/ml), and for bozacin B14 (no adsorption) (Ivanova, Kabadjova, Pantev, Danova, & Dousset, 2000; Todorov & Dicks, 2005). 3.5. Determination of approximate molecular weight of bacteriocins The molecular weight of the bacteriocins SD1, SD2, SD3 and SD4 was found to be approximately 5 kDa, as determined by tricineSDS-PAGE (Fig. 4), and it is within the range of most bacteriocins reported for other E. faecium (De Vuyst & Vandamme, 1994, p. 539). 3.6. Effect of temperature, pH and chemicals on bacteriocins adsorption to L. monocytogenes 211 Treatment of the cell-free supernatants with the proteolytic enzymes tested resulted in complete inactivation of antimicrobial activity (Table 2), whereas neither catalase nor a-amylase and lipase affected the activity, discarding clearly the involvement of

315

H2O2 in the antagonism process and suggesting that all these bacteriocins do not belong to the controversial group IV, which contain carbohydrates or lipids in the active molecule (De Vuyst & Vandamme, 1994, p. 539; Lewus, Sun, & Montville, 1992). Bacteriocins activities were not affected by salts and surfactants used (Table 2). The sensitivity to detergents, NaCl and urea seems to be bacteriocin-dependent and their stability may be affected by the experimental conditions during the purification or application processes. The four bacteriocins were not affected by the pH as they remained active after incubation for 2 h at pH ranging from 2.0 to 12.0 (Table 2). In some cases loss of activity at this extreme pH has been reported and ascribed to proteolytic degradation, protein aggregation or instability (De Vuyst, Callewaert, & Crabbe, 1996; Parente & Ricciardi, 1994). Bacteriocins SD1 and SD2 remained stable after 2 h at 60, 70, 80, 90 and 100
C (Table 2). Slight decrease in activity was observed upon heat treatment at 121
C for 15 min at pH 6.0 (data not shown). Bacteriocin SD3 was inactivated after exposure to 121
C

Fig. 4. SDS-PAGE of bacteriocin SD1 (A), SD2 (B), SD3 (C) and SD4 (D). The gel was covered with viable cells of L. monocytogens 211 (approx. 106 CFU/ml), imbedded in BHI supplemented with 1% agar.

316

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Table 2 Effect of temperatures, pH, chemicals, enzymes on the antimicrobial activity of the selected strains on the reference strain L. monocytogenes 211. Activity of bacteriocin is expressed in: (þ) ¼ presence of inhibition zone (>2 mm); () ¼ no inhibition. Bacteriocins

Enzymes a-amylase, catalase, lipase papain, trypsin, pepsin pronase, proteinase K Detergents/chemicals (1%) Laourylsarcosine NaCl SDS Triton X-100 Tween 20 Tween 80 Urea Temperatures 60, 70, 80 and 90
C for up to 120 min 100
C for up to 120 min 121
C for 15 min pH 2-12

SD1

SD2

SD3

SD4

þ e e

þ e e

þ e e

þ e e

þ þ þ þ þ þ þ

þ þ þ þ þ þ þ

þ þ þ þ þ þ þ

þ þ þ þ þ þ þ

þ

þ

þ

þ

þ þ

þ þ

þ e

e e

þ

þ

þ

þ

for 15 min, whilst bacteriocin SD4 loses its activity after 40 min at 100
C. Similar results were recorded for pediocins and other enterocins (Bhunia, Kim, Johnson, & Ray, 1988; Foulquié Moreno, Callawaert, Devreese, Van Beeumen, & De Vuyst, 2003).

3.7. Effect of bacteriocins on L. monocytogenes strains Cell free supernatants obtained from 24-h-old cultures of E. faecium strains were added to 3-h-old cultures of L. monocytogenes 211 serotype 4b (OD600nm z 0.06) over 12 h (Fig. 5). The bacteriocin secreted by E. faecium SD4 only produced a 50% growth inhibition on L. monocytogenes 211 after 12 h growth, as compared to the untreated (control) culture (Fig. 5C). On the other hand, the SD1, SD2 and SD3 supernatants resulted in a complete growth repression of the L. monocytogenes 211 test strain (no viable cells recorded after 12 h) (Fig. 5C). This suggests a bactericidal mode of activity of these bacteriocins. It is important to underline that these experiments were performed with high listerial initial cell numbers (approximately 108 CFU/ml), while lower cell numbers are expected in spoiled food, indicating that these bacteriocins could be reliable tools to control listerial food contamination. When early stationary phase cells of L. monocytogenes 211 were exposed to bacteriocins SD1, SD2, SD3 no viable cells were detected after incubation at 37
C for 1 h, while for SD4 the number was only reduced from 5.7  108 CFU/ml to 3.4  105 CFU/ ml, which is considered insufficient for applications in food biopreservation. Similar results were reported for bacteriocin ST44AM which, after 1 h of contact, completely inhibited the growth of stationary phase cells of Listeria ivanovii subsp. ivanovii ATCC 19119, Listeria innocua 2030C and E. faecium HKLHS (Todorov & Dicks, 2009a).

Fig. 5. Effect of bacteriocins SD1, SD2, SD3 and SD4 on (A) L. monocytogenes 637, (B) L. monocytogenes 620 and (C) L. monocytogenes 211. The arrow indicates the point at which the bacteriocins were added.

S. Schirru et al. / Food Control 25 (2012) 309e320

The addition of 51,200 AU/ml bacteriocins SD1 and SD2 and 3200 AU/ml of bacteriocin SD3 to a 3-h-old culture of L. monocytogenes 211, L. monocytogenes 620 and L. monocytogenes 637 (OD600nm ¼ 0.1) resulted in growth inhibition after 12 h exposure, as the addition of 800 AU/ml of bacteriocin SD4 resulted in not sufficient growth inhibition, indicating that its mode of activity is rather bacteriostatic (Fig. 5). Addition of the same level of the four bacteriocins to the stationary-phase cells target strains cultures, resulted in no significant growth inhibition (data not shown). This finding suggests that the tested bacteriocins are only active against actively growing cells. Bacteriocins were also evaluated also for their absorption to several target strains (Table 3). The highest level of absorption for bacteriocin SD1 and SD2 (57.14%) was recorded for L. monocytogenes 637 and 620 respectively, while for bacteriocin SD3 for L. monocytogenes 620 (71.43%). Finally bacteriocin SD4 was adsorbed at 33% to L. monocytogenes 211, 620 and 637 and L. sakei ATCC 15521 and at 66.67% to E. faecium ATCC 19443 (Table 3). In literature varying levels of adsorption (from 17 to 100%) are reported for different strains, but in general highest levels were detected for sensitive strains as compared to resistant strains (Todorov, Botes, Danova, & Dicks, 2007; Todorov & Dicks, 2006; Yildirim et al., 2002). In Table 3, the absorption levels of bacteriocins to the test strain L. monocytogenes 211 are also reported in relation to different pHs. Optimal adsorption of bacteriocin SD1 (57.14%) was exhibited at pH 8.0 and 10.0, as lower levels of pH (6.0 and 4.0) resulted in a reduction of the adsorption. For bacteriocin SD2 optimal adsorption (57.14%) was detected at pH 10.0 and lower at pH 8.0, 6.0 and 4.0. Optimal adsorption of bacteriocin SD3 (71.43%) was observed at pH 4.0, whereas higher levels of pH resulted in a clear reduction. For bacteriocin SD4 a value of 66.67% was obtained at pH 4.0 and progressively reduced at higher pH (6.0, 8.0 and 10.0). These results indicate a possible application potential of the selected bacteriocins at different pH ranges, as bacteriocins SD1 and SD2 may be applied in neutral to basic pH level products,

Table 3 Effect of temperature, pH and chemicals on adsorption of bacteriocins SD1, SD2, SD3 and SD4 to target microrganisms. Adsorption (%) of bacteriocin produced by Enterococcus faecium

L. monocytogenes 4b, 211 L. monocytogenes 4b, 620 L. monocytogenes 1/2c, 637 E. faecium ATCC 19443 L. sakei ATCC 15521

SD1

SD2

42.86 42.86 57.14 42.86 28.57

42.86 57.14 42.86 42.86 57.14

SD3 57.14 71.43 57.14 42.86 57.14

SD4 33.33 33.33 33.33 66.67 33.33

Adsorption (%) of bacteriocin produced by Enterococcus faecium strains to L. monocytogenes 211 Temperatures (
C) 4 25 30 37 pH 4.0 6.0 8.0 10.0 Chemicals (1%) Tween 80 Tween 20 SDS NaCl Na2HPO4 NaH2PO4

42.86 57.14 42.86 57.14

28.57 28.57 42.86 42.86

100 71.43 57.14 42.86

66.67 66.67 33.33 66.67

42.86 42.86 57.14 57.14

28.57 42.86 42.86 57.14

71.43 57.14 57.14 42.86

66.67 33.33 33.33 0

57.14 57.14 0 42.86 57.14 57.14

57.14 42.86 14.29 14.29 57.14 42.86

71.43 57.14 71.43 57.14 71.43 85.71

66.67 66.67 100 33.33 33.33 33.33

317

Table 4 Effect of antibiotics on the growth of Enterococcus faecium strains SD1, SD2, SD3 and SD4. The values represent the diameter of the inhibition zone. Antibiotic (mg/disk)

Inhibition zone (mm) SD1

SD2

SD3

SD4

Cefazolin (30) Cefalotin (30) Cefotaxim (30) Cefoxitin (30) Cfaclor (30) Doxycyclin (30) Minocyclin (30) Ceftazidim (30) Ceftriaxon (30) Ceftiofur (30) Moxifloxacin (5) Cefepim (30) Ampicillin þ sulbactam (20) Cotrimazin (25) Cotrimixazol (25) Tazobactam (10) Amicacin (30) Gentamycin (10) Kanamycin (30) Neomicin (30) Streptomycin (10) Tobramycin (10) Spectinomycin (10) Streptomicin (300) Chloramphenicol (30) Florphenicol (30) Furazolidon (10) Imipenem (10) Amoxicillin þ clavulanic acid (30) Bacitracin (10) Ciprofloxacin (5) Eurofloxacyn (5) Vancomycin (30) Clindamycin (2) Ofloxacin (5) Clarithromycin (15) Erythromycin (15) Tilmicosin (15) Nitrofurantoin (300) Linezolid (30) Penicillin G (10) Ampicillin (10) Rifampicin (30) Rifampicin (5) Sulphonamid (300) Nalidixic acid (30) Tetracyclin (30) Cefuroxim (30) Oxacillin (1)

17 0 23 15 24 32 34 13 22 22 25 21 27 0 0 27 25 22 25 23 18 20 23 27 30 34 12 29 28

15 22 21 19 24 33 36 12 20 0 21 20 26 0 0 0 26 22 21 22 14 20 20 22 32 29 15 25 0

19 0 24 20 19 30 35 13 20 25 18 23 24 0 0 27 29 27 28 25 22 20 22 29 30 29 17 23 25

16 0 16 18 20 36 39 15 15 0 23 0 0 0 0 29 12 14 17 20 13 12 20 25 36 33 12 27 28

31 18 13 22 35 0 30 31 20 24 29 22 26 26 29 0 0 30 18 0

29 18 18 22 31 17 35 33 22 21 30 24 30 34 24 0 0 33 24 0

31 18 20 24 32 19 30 32 21 0 25 31 30 27 27 0 0 28 26 0

24 20 21 20 34 22 30 36 15 26 29 34 27 32 19 0 0 32 14 0

and bacteriocins SD3 and SD4 may have application in products having lower pH. The different levels of adsorption observed by varying the pH may be due to specific interaction between the four bacteriocins and the target strain. In the same Table 3 the effect of temperature is also reported. The maximum adsorption for bacteriocin SD1 was recorded at 25
C and 37
C (57.14%), while it decreased at 4
C and 30
C (42.86%). For bacteriocin SD2, 30
C and 37
C resulted in the highest absorption values (42.86%), clearly reduced at 4
C and 25
C (28.57%). Levels of 100% adsorption was obtained at 4
C for bacteriocin SD3 and a progressive decrease was recorded increasing the temperature. Optimal adsorption for bacteriocin SD4 (66.67%) was observed at 4
C, 25
C and 37
C. These findings showing that temperature can affect differently the adsorption of bacteriocins to target strains are in agreement with data reported in literature (Todorov & Dicks, 2006; Yildirim et al., 2002).

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No adsorption of bacteriocin SD1 to L. monocytogenes 211 was observed in the presence of SDS, 42.86% with NaCl and 57.14% once treated with Tween 80, Tween 20, Na2HPO4 and NaH2PO4. Adsorption levels of 57.14% were detected for bacteriocin SD2 after the treatment with Tween 80 and Na2HPO4, while Tween 20 and NaH2PO4 produced 42.86% adsorption and SDS and NaCl treatments resulted in only 14.29%. For bacteriocin SD3 highest levels of adsorption (85.71%) were detected once treated with NaH2PO4 and lower levels with NaCl and Tween 20. Different results were observed for bacteriocin SD4, showing 100% adsorption in the presence of SDS and only 33.33% with NaCl, Na2HPO4 and NaH2PO4 (Table 3). The results of this study, according to previous data reported in literature, clearly confirm that the influence of environmental parameters (pH, temperature) as well as chemical compounds on adsorption is bacteriocin-specific (Manca de Nadra, Sandino de Lamelas, & Strasser de Saad, 1998; Nieto-Lozano, Reguera-Useros, Pelaez-Martinez, & De la Torre, 2002). 3.8. Growth at different pH values and NaCl concentrations Once grown in MRS with pH adjusted between 2 and 10, all test strains exhibited growth curves as expected by Enterococcus spp. and, more widely, from LAB (data not shown). At pH below 5.0, the strains were strongly inhibited and at pH 10 their growth was much slower than that showed in the control MRS medium. At pH values higher than that of the control broth (pH 7e9), their growth was more rapid. Increasing amounts of sodium chloride in the MRS broth also influenced microbial growth. E. faecium SD1 grew well up to 2% NaCl and its growth showed a slight increase after 10 h at higher salt concentrations (data not shown). Strain SD2 was able to grow in MRS containing up to 5% sodium chloride (data not shown). This pattern is similar to that shown by SD3, with the exception that some adaptation to the highest NaCl concentration occurred after prolonged incubation. In contrast, the presence in the broth of 4% NaCl was high enough to slow down the microbial growth of E. faecium SD4. According to the results obtain from the growth of SD1, SD2, SD3 and SD4 in presence of different pH values and NaCl concentrations, the physiological traits of the strains seem to be suitable for the production of several artisanal and traditional cheeses and in particular, E. faecium SD2 and SD3 may be utilized in Pecorino-like cheeses processing where sodium chloride content is higher than 3% (De Angelis et al., 2001; Foulquié Moreno et al., 2006; Suzzi et al., 2000). 3.9. Technological characterization of the selected strains E. faecium strains were evaluated for acidifying, lipolytic and proteolytic activities. The acidifying activity was generally low: 5.80  0.13 for SD1, 5.66  0.15 for SD2, 5.13  0.13 for SD3 and 5.59  0.12 for SD4 after 24 h incubation in milk with initial pH of 6.45; 4.70  0.08 for SD1, 4.67  0.11 for SD2, 4.73  0.09 for SD3 and 4.75  0.10 for SD4 after 24 h incubation in MRS broth with initial pH of 6.38; 5.88  0.11 for SD1, 5.88  0.16 for SD2, 5.89  0.10 for SD3 and 5.86  0.12 for SD4 after 24 h incubation in BHI broth with initial pH of 7.40, suggesting their possible role as adjunct cultures for cheese production, rather than as starter micro-organisms. A good acid-producing starter culture will reduce the pH of milk from its normal value of about 6.6 to 5.3 in 6 h using an inoculum of 10% and in general, enterococci exhibit low milk acidifying ability (Cogan et al., 1997). Investigations on enterococci of dairy origin confirmed the poor acidifying capacity of these microbes in milk with only a small percentage of the strains

showing a pH below 5.0e5.2 after 16e24 h of incubation at 37
C (Giraffa, 2003). Enterococci occur as NSLAB (non-starter LAB) in various, especially artisanal, cheeses produced in the southern Europe from goat, ewe, water buffalo or bovine milk. Since enterococci may dominate the NSLAB of many cheeses, it is supposed that they can positively contribute to the flavor development during cheese ripening. As a consequence, enterococci may improve the sensory characteristics of the final product (Cogan et al., 1997; Comunian et al., 2010; De Angelis et al., 2001). Lipolysis is also important in cheese ripening as it plays a role in the development of flavor and texture of the ripened product. Very few data, on the lipolytic activity of enterococci has been previously reported, with E. faecalis being the most lipolytic species, followed by E. faecium and Enterococcus durans (Giraffa, 2003; Madrau et al., 2006). Results obtained in this study strengthened the view that the species E. faecium generally has low lipolytic activity. Indeed, no strain gave positive results on trybutirin agar medium (data not shown). Protease activity is necessary for good growth of LAB in milk and for casein hydrolysis during cheese ripening. In this perspective, conflicting literature data concerning casein proteolysis in enterococci indicate a marked strain-to-strain variation of this phenotypic feature (Giraffa, 2003). The results of the present study confirmed these findings. Indeed, among the four E. faecium strains, only SD3 and SD4, showing low enzymatic activities, could be considered weak proteolytic isolates (with OPA values 0.09  0.01 for SD3 and 0.06  0.01 for SD4). However, their proteolytic activity, was similar to those produced by several LAB strains described in Madrau et al. (2006). 3.10. Susceptibility of the selected strains to antibiotics The majority of the tested antibiotics, selected for their variety of action mechanisms, inhibited to some extent the growth of the strains under study (Table 4). Only five of them (cotrimazin, cotrimixazol, nalidixic acid, oxacilin and sulphonamid) had no inhibitory effect on the strains isolated. In addition, E. faecium SD1 was resistant to cefalotin and oflaxacin, the isolate SD2 to amoxicillin þ clavulanic acid, ceftiofur and tazobactam. The strain SD3 was not inhibited by cefalotin and nitrofurantion whereas E. faecium SD4 by ampicillin þ sulbactam, cefalotin, cefpime and ceftiofur. Resistance of LAB to antibiotics is a controversial subject. It is important to keep in mind that potential probiotic LAB may be reservoir of antibiotic resistance genes, and horizontal gene transfer to the other bacteria present in the human GIT is possible (Dicks, Todorov, & Franco, 2009). The large release of antibiotics in the biosphere, more than 10 million tons, according to the European Commission (2005), is responsible for selection of antibiotic resistant strains. Resistance may be inherent to a bacterial genus or species, but may also be acquired through exchange of genetic material, mutations and the incorporation of new genes (Ammor, Flóres, & Mayo, 2007). Teuber (1999) suggested that starter cultures and probiotics may serve as vectors in the transfer of antibiotic resistant genes. Among LAB, enterococci are frequently associated with antimicrobial resistance markers and for this reason, a number of studies have attempted to compare the resistance spectra of different enterococci according to their human, animal or food origins. However, although antibiotic resistances have been detected in foods, resistance to the clinically relevant antibiotics such as ampicillin, penicillin, gentamycin and vancomycin occurs in very few isolates (Comunian, 2010; Mannu et al., 2003). In a previous study on Fiore Sardo PDO cheese it was reported that isolates harboring virulence or antibiotic resistance

S. Schirru et al. / Food Control 25 (2012) 309e320

genes were at so low presence to be not considered as contributing to efficient horizontal genes transfer in the GIT (Comunian et al., 2010). Therefore, the presence of enterococci in Fiore Sardo cheese should not represent a threat to human health. 4. Conclusions Enteroccocci are commonly and abundantly found in a variety of cheeses produced in Mediterranean part of Europe from raw goats’, ewes’, water buffalo or cows’ milk. In many traditional cheeses enterococci are important in ripening and aroma development. Based on previously reviewed and our results, the selected E. faecium SD1, SD2, SD3, SD4 strains could be considered as suitable for use as adjunct or protective cultures in cheese production. Moreover, the bacteriocin producers evaluated in this study may have potential applications in the food and feed industry as effective biopreservatives. However a future study on presence of virulence factors and biogenic amine formation needs to be examined in detail in order to prove safety of these strains. Acknowledgments Dr. Schirru was supported by Agris - Agricultural Research Agency of Sardinia (Italy), Dr. Todorov was supported by CAPES and CNPq, Brasilia, DF (Brazil), Dr. Favaro was recipient of “Assegno di ricerca Junior” grant from University of Padova (Italy). Authors are grateful to Prof. Maria Teresa Destro and Dr. Eb Chiarini (University of Sao Paulo, Sao Paulo, Brazil) for providing the Listeria monocytogenes strains used in this study and to Dr. Salvatore Marceddu (University of Sassari, Italy) for performing SEM. References Ammor, M. S., Flóres, A. B., & Mayo, B. (2007). Antibiotic resistance in notenterococcal lactic acid bacteria and bifidobacteria. Food Microbiology, 24, 559e570. Ananou, S., Gálvez, A., Martýnez-Bueno, M., Maqueda, M., & Valdivia, E. (2005). Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli 0157:H7. Journal of Applied Microbiology, 99, 1364e1372. Bhunia, A. K., Kim, W. J., Johnson, M. S., & Ray, B. (1988). Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. Journal of Applied Bacteriology, 65, 261e268. Bozzola, J. J. (2007). Conventional specimen preparation techniques for transmission electron microscopy of cultured cells. In J. Kuo (Ed.), Methods in molecular biologyÔ. Electron microscopy methods and protocols, Vol. 369 (pp. 1e18). Totowa, New Jersey: Humana Press Inc. Bridge, P. D., & Sneath, P. H. A. (1983). Numerical taxonomy of Streptococcus. Journal of General Microbiology, 129, 565e597. Charoenchai, C., Fleet, G. H., Henschke, P. A., & Todd, B. (1997). Screening of nonSaccharomyces wine yeasts for the presence of extracellular hydrolytic enzymes. Australian Journal of Grape and Wine Research, 3, 2e8. Chen, H., & Hoover, D. G. (2003). Bacteriocins and their food applications. Comprehensive Reviews in Food Science and Food Safety, 2, 81e100. Church, F. C., Swaisgood, H. E., Porter, D. H., & Catignani, G. L. (1983). Spectrophotometric assay using o-Phthaldialdehyde for determination of proteolysis in milk and isolated milk proteins. Journal of Dairy Science, 66, 1219e1227. Cogan, T. M., Barbosa, M., Beuvier, E., Bianchi-Salvadori, M. B., Cocconcelli, P. S., Fernandes, I., et al. (1997). Characterization of the lactic acid bacteria in artisanal dairy products. Journal of Dairy Research, 64, 409e421. Comunian, R. (2010). Identification and safety assessment of enterococci isolated from a Sardinian ewe’s raw milk PDO cheese (Fiore Sardo). PhD Thesis, Facoltà di Medicina Veterinaria, Università di Sassari. Publicly defended in Sassari, February 1st, 2010. Comunian, R., Daga, E., Dupré, I., Paba, A., Devirgiliis, C., Piccioni, V., et al. (2010). Susceptibility to tetracycline and erythromycin of Lactobacillus paracasei strains isolated from traditional Italian fermented foods. International Journal of Food Microbiology, 138, 151e156. De Angelis, M., Corsetti, A., Tosti, N., Rossi, J., Corbo, M. R., & Gobbetti, M. (2001). Characterization of non-starter lactic acid bacteria from Italian ewe cheeses based on phenotypic, genotypic, and cell wall protein analyses. Applied Environmental Microbiology, 67, 2011e2020. De Kwaadsteniet, M., Todorov, S. D., Knoetze, H., & Dicks, L. M. T. (2005). Characterization of a 3944 Da bacteriocin, produced by Enterococcus mundtii ST15,

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