Comparative antimicrobial activity of enterocin L50, pediocin PA1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria

Food Microbiology, 1998, 15, 289-298

ORIGINAL ARTICLE

Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria ´ ´ L. M. Cintas*, P. Casaus, M. F. Fernandez and P. E. Hernandez

The present work compares, under the same stated experimental conditions, the antimicrobial activity of crude and purified enterocin L50, pediocin PA-1, nisin A and lactocin S, produced by lactic acid bacteria (LAB) isolated from Spanish dry-fermented sausages. The bacteriocins were purified to homogeneity by ammonium sulphate precipitation, gel filtration (for lactocin S), and cation-exchange, hydrophobic-interaction, and reverse-phasechromatography; high yields of pure bacteriocins were obtained. Minimal inhibitory concentration (MIC) of pure enterocin L50, pediocin PA-1, nisin A and lactocin S was determined against a broad spectrum of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The purified bacteriocins showed a broad antimicrobial spectrum similar to that exerted by crude bacteriocins. Enterocin L50 and pediocin PA-1 were very active against Listeria monocytogenes, which was quite resistant to nisin A and lactocin S. Enterocin L50 also displayed antimicrobial activity against Staphylococcus aureus, Clostridium perfringens and Clostridium botulinum. However, these pathogens were weakly inhibited, or not at all, by the other pure bacteriocins.  1998 Academic Press

Introduction Lactic acid bacteria (LAB) are recognized to play an important role in food fermentations and food preservation, either as the natural microflora or as a starter culture added under controlled conditions. Lactococcus, Lactobacillus, Leuconostoc and Pediococcus are the genera most commonly used as starter cultures in fermentation processes of milk, meat and vegetable products (Liepe 1983, Stiles and Hastings 1991). These organisms ensure and enhance the development of the desired changes in texture, flav*Corresponding author. 0740-0020/98/030289+10 $30·00/0/fd970160

our, colour, digestibility and nutritional qualities of the fermented products (Smith and ¨ Palumbo 1981, Schillinger and Lucke 1987, 1989). Besides their technological roles, LAB also increase the hygiene quality and safety of foods and food products by inhibiting the natural competing flora, which includes spoilage and pathogenic bacteria (Raccah et al. 1979, Smith and Palumbo 1983, Klaenhammer ¨ 1988, Daeschel 1989, Schillinger and Lucke 1990). The primary antimicrobial effect exerted by LAB is production of lactic acid and reduction of pH (Daeschel 1989). However, they also produce a number of inhibitory compounds other than organic acids, which include hydrogen peroxide, CO2, diacetyl, acetaldehyde, D-isomers of amino

Received: 27 July 1997 Departamento de ´ Nutricion y ´ Bromatologıa III, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040Madrid, Spain

 1998 Academic Press

290 L. M. Cintas et al.

acids, and bacteriocins (Klaenhammer 1988, Daeschel 1989, Stiles and Hastings 1991, Piard and Desmazeaud 1991, 1992). Bacteriocins are a heterogeneous group of ribosomally synthesized peptides displaying antimicrobial activity against other bacteria (Klaenhammer 1993). Three well-defined classes of bacteriocins in LAB have been established: class I, the lantibiotics; class II, the small, heat-stable non-lantibiotics; and class III, the large heat-labile bacteriocins (Nes et al. 1996). Several reports reviewing LAB bacteriocins have been published recently (Hoover and Steenson 1993, Klaenhammer 1993, Nettles and Barefoot 1993, de Vuyst and Vandamme 1994, Dodd and Gasson 1994, Jack et al. 1995, Nes et al. 1996). Concerning the use of bacteriocins and/or micro-organisms as natural preserving agents in food processing, we have characterized several bacteriocins produced by LAB isolated from Spanish dry-fermented sausages. These bacteriocins include enterocin L50, produced by Enterococcus faecium L50 (previously identified as Pediococcus acidilactici L50 [Cintas 1995, Cintas et al. 1995]), pediocin PA-1, produced ´ by P. acidilactici Z102 (Cintas 1995, Rodrıguez et al. 1997), nisin A, produced by Lactococcus lactis subsp. lactis BB24 (L. lactis ´ BB24) and L. lactis G18 (Cintas 1995, Rodrıguez et al. 1995), lactocin S, produced by Lactobacillus sake V18 and Lb. sake 148 (Sobrino et al. 1992, Cintas 1995), enterocins A and B, produced by E. faecium T136 (Casaus et al. 1995, 1997), and enterocin P, produced by E. faecium P13 (Cintas et al. 1997). The present work represents, to our knowledge, the first comparative study, under the same stated experimental conditions, of thoroughly characterized antimicrobial activities of crude and pure nisin A (Hurst 1981, Dodd et al. 1990, Harris et al. 1992, Siegers and Entian 1995), lactocin S (Mortvedt and Nes 1990, Mortvedt et al. 1991, Skaugen et al. 1997), and pediocin PA´ 1 (Gonzalez and Kunka 1987, Pucci et al. 1988, Henderson et al. 1992, Nieto-Lozano et al. 1992, Chikindas et al. 1993). This study also includes the antimicrobial activity of enterocin L50, a new small and heat-stable non-lantibiotic peptide bacteriocin from E.

faecium L50, which does not belong to the pediocin family of bacteriocins and shows a broad antimicrobial spectrum (Cintas 1995, Cintas et al. 1995).

Materials and methods Bacterial strains and media The bacteriocinogenic strains were previously isolated in our laboratory from four different Spanish dry-fermented sausages, manufactured with no added starter cultures (Cintas 1995, Cintas et al. 1995). The pediocin PA-1-, nisin A-, and lacto S-producing micro-organisms were P. acidilactici Z102, L. lactis BB24, and Lb. sake V18, respectively. Enterocin L50 is the same bacteriocin to that previously referred to as pediocin L50 from P. acidilactici L50. Total proteins of strain L50 were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDSPAGE) (Laemmli 1970), and the strain was further submitted to the tests proposed by ¨ Schleifer and Kilpper-Balz (1984) and Devriese et al. (1993). The strain L50 was reclassified as Enterococcus faecium L50, and consequently by the bacteriocin renamed enterocin L50. The Gram-positive bacteria used as indicators are shown in Table 1. The LAB were propagated in MRS broth (Oxoid Ltd, Basingstoke, UK) at 32°C or 37°C. Clostridium spp. strains were propagated anaerobically (Oxoid Anaerobic System) in RCM broth (Oxoid) at 32°C or 37°C. Propionibacterium spp. strains were grown anaerobically in GYE medium (Cintas et al. 1995) at 32°C. All other strains were propagated in BHI broth (Oxoid) at 32°C or 37°C.

Preparation of bacteriocin extracts Cell-free supernatants and 20-fold-concentrated cell-free supernatants (crude bacteriocins) from E. faecium L50, P. acidilactici Z102, L. lactis BB24 and Lb sake V18 were prepared essentially as described by Cintas et al. (1995). Briefly, bacteriocin-producing strains were grown in MRS broth at 32°C until early stationary phase. Cultures were subsequently centrifuged at 12 000 g for

Comparative activity of bacteriocins 291

10 min at 4°C. To eliminate growth inhibition caused by low pH-value and hydrogen peroxide, the pH of the supernatants was neutralized to 6·2 with 1 M NaOH, and 130 U of catalase (Boehringer GmbH; Mannheim, Germany) per ml were added. The supernatants were filter-sterilized through 0·22µm pore-size filters (Millipore Corp., Bedford, Massachussetts). Concentrated supernatants were prepared by lyophilization. The lyophilized samples were dissolved to 1/20 of the original volume in 4 mM phosphate buffer (pH 7·0). The bacteriocin extracts were stored at −20°C until further use.

Bacteriocin assays The antimicrobial activity of supernatants and 20-fold-concentrated supernatants was evaluated by an agar well-diffusion test (Cintas et al. 1995), against the selected indicator micro-organisms listed in Table 1. After growth of the indicator micro-organisms, the antimicrobial activity was determined by measuring the diameter (mm) of the inhibition zones. The bacteriocin activity during the purification processes was quantified in a microtitre plate assay (Holo et al. 1991). Briefly, two-fold serial dilutions (50 µl) of bacteriocin extracts in MRS broth were prepared in microtitre plates. The wells were then filled up to 200 µl by the addition of 150 µl of a fresh diluted overnight culture of P. pentosaceus FBB61 (A620:0·1). Growth inhibition of indicator strain was measured spectrophotometrically at 620 nm by using a microtitre plate reader (Titertek Multiskan Plus; Flow Laboratories, Helsinki, Finland) after 14 h incubation at 32°C. One arbitrary bacteriocin unit (BU) was defined as the reciprocal of the highest dilution of bacteriocin which inhibited the growth of the indicator microorganism by 50% (50% of the turbidity of the control culture without bacteriocin) under stated assay conditions.

Purification of bacteriocins Purification of enterocin L50, pediocin PA-1, nisin A, and lactocin S was achieved using a modification of the procedure described by

Nissen-Meyer et al. (1992). All the chromatographic equipment was obtained from Pharmacia-LKB (Uppsala, Sweden) and all the purification steps were performed at room temperature if not otherwise stated. (i) Ammonium sulphate precipitation. The bacteriocins were purified from 1-litre culture of the bacteriocinogenic strains grown in MRS broth at 32°C until early stationary phase. The cells were removed by centrifugation at 12 000 g for 10 min at 4°C, and ammonium sulphate was gradually added to achieve 75% saturation for enterocin L50, pediocin PA-1 and nisin A, and 45% saturation for lactocin S. The samples were kept at 4°C with stirring for 30 min, and then centrifuged at 12 000 g for 20 min at 4°C. The pellets and floating solid material were combined and solubilized in buffer A (200 ml of 20 mM sodium phosphate, pH 5·8, for enterocin L50, pediocin PA-1 and nisin A; 50 ml of 5 mM sodium phosphate, pH 5·0 for lactocin S). (ii) Gel filtration. The fraction containing lactocin S was passed through Sephadex G25 PD-10 columns, before application to the cation-exchange column, in order to remove any trace of ammonium sulphate. The columns were then equilibrated with 5 mM sodium phosphate, pH 5·0, and the bacteriocin was recovered in the same buffer. (iii) Cation-exchange chromatography. Fractions with bacteriocin activity were then applied to an 8 ml (15 ml for nisin A) SPSepharose Fast Flow cation-exchange column equilibrated with buffer A. After washing the column with 50 or 100 ml of buffer A, the bacteriocin activity was eluted with the same volume of 1 M NaCl in buffer A. (iv) Hydrophobic interaction chromatography. Ammonium sulphate was added, to a final concentration of 10% (w/v), to the fractions eluted from the cation-exchanger. The samples were subsequently applied to a 3-ml Octyl-Sepharose CL-4B column equilibrated with buffer A containing ammonium sulphate (10% w/v). After washing the column with 15 ml of the equilibrium buffer, the bacteriocin activity was eluted with 10 ml of buffer A containing 50% (v/v) ethanol. (v) Reverse-phase chromatography. Trifluoroacetic acid (TFA, Merck) was added, to

292 L. M. Cintas et al.

a final concentration of 0·1% (v/v), to the fractions eluted from the Octyl-Sepharose column. Fractions were filtered through 0·22µm pore-size filters (Millipore), five-fold diluted in aqueous 0·1% (v/v) TFA (buffer B), and subsequently applied, at a flow rate of 1 ml min−1, to a C2/C18 reverse-phase column, PepRPC HR5/5, integrated in an FPLC System and equilibrated with buffer B. The bacteriocin activity was then eluted from the column with a linear gradient of 2-propanol (Merck) in buffer B, at a flow rate of 0·5 ml min−1. Fractions with bacteriocin activity were made up to 10 ml with buffer B, and directly rechromatographed on the same reverse-phase column, under the same conditions, in order to get chromatographically pure bacteriocins. Purified enterocin L50, pediocin PA-1, nisin A and lactocin S were stored in 50–60% 2-propanol containing 0·1% (v/v) TFA at −20°C. Concentration of enterocin L50, pediocin PA-1, nisin A and lactocin S in fractions from the last reversephase chromatography step was determined using the Coomassie Protein Assay Reagent kit (Pierce Chemicals, Rockford, Illinois), according to the suppliers’ instructions.

MIC of enterocin L50, pediocin PA-1, nisin A and lactocin S MIC of purified bacteriocins was determined by the microtitre plate assay, with adequate growth media and incubation conditions for each indicator strain listed in Table 3, as described by Cintas et al. (1995). Briefly plates containing two-fold serial dilutions of a sample with a known amount of pure bacteriocin and the indicator micro-organism were prepared as described above, and incubated until the cultures with no added bacteriocin had reached the stationary phase. The MIC was defined as the bacteriocin concentration (ng ml−1) causing 50% growth inhibition.

Results Antimicrobial activity spectrum of crude enterocin L50, pediocin PA-1, nisin A and lactocin S The antimicrobial activity of supernatants

and 20-fold concentrated supernatants of the four bacteriocinogenic strains tested in simultaneous experiments is summarized in Table 1. The growth of most LAB was inhibited by both the supernatants and the concentrated supernatants of the four strains. E. faecalis, Staphylococcus carnosus and Listeria innocua were also inhibited by supernatants from E. faecium L50, P. acidilactici Z102 and Lb. sake V18, but they were weakly inhibited by supernatants from nisinproducing Lactococcus lactis BB24. Bacillus cereus ATCC9139 was the only micro-organism not inhibited by any of the crude bacteriocins. However, the growth of the spoilage bacteria Clostridium sporogenes, Cl. tyrobutyricum and Propionibacterium spp. was affected by the supernatants from the four bacteriocin-producing strains. Both supernatants and concentrated supernatants of either P. acidilactici Z102, L. lactis BB24 or Lb. sake V18 inhibited the growth of the foodborne pathogens Cl. perfringens and Cl. botulinum, while only the concentrated supernatant from E. faecium L50 was active against these pathogens. Listeria monocytogenes and S. aureus strains were inhibited by supernatants from the four bacteriocin-producing strains, although the inhibition produced by Lactococcus lactis BB24 and Lb. sake V18 extracts was much weaker than that obtained by the extracts from P. acidilactici Z102 and E. faecium L50.

Purification of enterocin L50, pediocin PA-1, nisin A and lactocin S Enterocin L50, pediocin PA-1, nisin A and lactocin S were purified to homogeneity. The purification results are summarized in Table 2. The purification of enterocin L50, pediocin PA-1, lactocin S and nisin A increased their specific activities approximately 1·1×105, 6× 104, 1·6×105, and 9×106-fold compared with the specific activities of culture supernatants, respectively. The yields of enterocin L50, pediocin PA-1, lactocin S and nisin A after the last reverse-phase chromatography step were approximately 80, 44, 26 and 23 000% of the initial activities, respectively. From the chromatograms in the last purification step,

Comparative activity of bacteriocins 293

showing a single activity peak coinciding with the protein absorbance peak, it was concluded that the four bacteriocins had been purified to homogeneity. The recoveries of

pure bacteriocins from a 1-litre culture of E. faecium L50, P. acidilactici Z102, Lactococcus lactis BB24 and Lb. sake V18 are listed in Table 2.

Table 1. Antimicrobial activity of four lactic acid bacteria of meat origin against selected indicator micro-organismsa Enterococcus Pediococcus faecium acidilactici L50 Z102 Indicator speciesb

Strain

Lactobacillus acidophilus 4356 bulgaricus 11842 casei 334 curvatus 2739 fermentum 9338 helveticus 15009 sake 2714 salivarius 2747 Lactococcus cremoris CNRZ117 lactis CNRZ148 lactis CNRZ150 Leuconostoc cremoris DB1275 Pediococcus pentosaceus FBB61 pentosaceus FBB63 pentosaceus PC1 Clostridium sporogenes C22/10 tyrobutyricum 3,5CT tyrobutyricum 1754 Enterococcus faecalis EF Listeria innocua BL86/26 Propionibacterium sp. P4 Propionibacterium sp. P6 Prop. acidipropionici 573 Staphylococcus carnosus MC1 Bacillus cereus 9139 Clostridium perfringens 376 botulinum 551 Listeria monocytogenes 7973 monocytogenes LI5sv1/2 monocytogenes 5105 monocytogenes LI1sv4 monocytogenes ScottA Staphylococcus aureus 137 aureus 196E aureus 349 aureus 361 aureus 472 a

Lactococcus Lactobacillus lactis sake BB24 V18

Sourcec

A

B

A

B

A

B

A

B

ATCC ATCC ATCC NCFB ATCC ATCC NCFB NCFB INRA INRA INRA TNO TNO TNO TNO TNO TNO NCDO TNO TNO TNO TNO NCDO TNO ATCC CECT CECT NCTC FVM NCTC FVM FVM FRI FRI FRI FRI FRI

13·4 8·7 13·5 NIZD 15·4 8·0 NIZD NIZD 7·3 11·4 7·0 8·0 14·2 12·5 7·4 13·0 NIZD NIZD 12·6 12·0 13·2 15·0 13·3 11·6 NIZD NIZD NIZD 10·6 11·6 10·7 11·0 11·3 11·6 11·3 11·6 12·3 12·0

17·8 11·4 17·5 7·7 18·1 13·0 8·3 NIZD 9·4 18·0 11·3 12·0 17·4 16·5 9·7 16·2 12·0 13·0 14·4 15·0 17·0 18·6 16·3 14·6 NIZD 11·7 11·2 14·4 15·0 14·6 14·6 15·1 14·0 14·0 16·0 17·0 14·0

NIZD 14·3 NIZD 12·0 15·3 14·0 15·0 NIZD NIZD NIZD NIZD NIZD 17·0 11·0 NIZD 16·5 15·0 NIZD 14·4 14·0 14·0 14·0 15·0 13·5 NIZD 14·0 15·0 14·7 14·4 14·4 14·4 15·6 14·6 14·0 NIZD 13·3 13·0

NIZD 19·0 NIZD 16·0 21·0 20·0 17·8 9·6 NIZD NIZD NIZD NIZD 24·0 16·6 NIZD 22·5 21·0 NIZD 18·0 17·0 19·0 18·5 19·5 17·4 NIZD 18·0 19·0 17·6 17·6 17·0 18·0 18·0 18·0 18·0 NIZD 17·0 16·4

21·0 15·7 17·4 10·5 7·2 16·0 12·0 12·5 17·5 NIZD NIZD 11·2 17·4 11·1 12·2 18·0 17·0 17·0 8·7 NIZD 8·7 7·8 8·0 NIZD NIZD 17·0 16·0 7·4 7·0 8·0 8·0 8·0 8·0 8·5 NIZD 8·2 NIZD

22·0 16·3 18·3 11·0 7·4 17·3 12·0 12·0 17·6 10·4 NIZD 13·0 17·6 11·1 12·0 21·7 20·0 19·0 11·0 7·0 12·0 9·0 11·5 8·0 NIZD 18·4 18·6 11·0 10·2 11·0 10·5 11·4 8·0 8·2 NIZD 11·0 11·3

7·4 10·2 NIZD 10·0 NIZD 8·0 15·2 7·2 13·6 NIZD NIZD 10·8 12·6 15·8 9·6 7·8 15·0 17·8 9·6 9·4 9·6 17·0 14·0 NIZD NIZD 7·0 7·6 10·0 10·6 9·0 10·6 9·0 NIZD NIZD NIZD NIZD NIZD

10·8 20·6 11·4 16·4 9·8 12·5 21·0 12·2 21·4 NIZD NIZD 18·6 16·4 22·0 17·8 13·0 20·4 21·0 13·4 13·8 17·4 20·0 19·0 12·0 NIZD 10·2 9·0 12·6 13·4 13·0 13·8 13·0 12·2 13·5 13·0 15·9 11·4

Diameter of inhibition zone in millimetres expressed as the means of the results obtained with two simultaneous experiments. A, Supernatant; B, 20-fold-concentrated supernatant; NIZD, no inhibition zone detected using 50 µl of supernatant or 20-fold-concentrated supernatant. b Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis subsp. cremoris are abbreviated as Lactobacillus bulgaricus and Lactococcus cremoris, respectively. ´ c Abbreviations: ATCC, American Type Culture Collection (Rockville, USA);` CECT, Coleccion ˜ Espanolade Cultivos Tipo (Valencia, Spain); INRA, Station de Recherches Laitieres (Jouy-en-Josas Cedex, France); FRI, Food Research Institute (Madison, USA); FVM, Facultad de Veterinaria (Madrid, Spain); NCDO and NCFB, National Collection of Dairy Organisms (Reading, UK); NCTC, National Collection of Type Cultures (London, UK); TNO, Nutrition and Food Research (Zeist, The Netherlands).

294 L. M. Cintas et al.

MIC values of enterocin L50, pediocin PA-1, nisin A and lactocin S The antimicrobial activity of the purified bacteriocins remained stable during storage (6 months at −20°C), and a microtitre plate assay was used to evaluate simultaneously their MICs against selected micro-organisms (Table 3). Most of the LAB tested were inhibited by the purified enterocin L50, with MICs ranging from 13 to 260 ng ml−1, but no inhibition was detected against Lb. salivarius NCFB2747. While purified pediocin PA-1 was not active against Lb. acidophilus ATCC4356, P. pentosaceus PC1, Leuconostoc mesenteroides subsp. cremoris DB1275 (Leuconostoc cremoris DB1275) or Lactococcus spp., pediocin PA-1 concentrations down to 0·7 and 3 ng ml−1 were sufficient to inhibit the growth of P. pentosaceus FBB61 and Lb. helveticus ATCC15009, respectively. Purified nisin A showed an inhibitory spectrum against a variety of LAB, with MICs ranging from 2 to approximately 1500 ng ml−1. Pure lactocin S was less active than the other bacteriocins against LAB. Enterocin L50 was very effective against B. cereus ATCC9139, with MIC of 187 ng ml−1, in contrast to the observation with supernatants. This foodborne pathogen was also inhibited by nisin A, lactocin S and pediocin PA-1, but at very high bacteriocin concentrations (778, 1990 and 3086 ng ml−1, respectively). The four purified bacteriocins inhibited growth of most of the spoilage bacteria tested, with enterocin L50 and pediocin PA-1 being the most effective compounds. Only 0·2 ng ml−1 of pediocin PA-1 were needed to inhibit growth of the spoilage bacteria Propionibacterium spp. On the other hand, the two Cl. tyrobutyricum strains tested were not sensitive to pure pediocin PA-1. Pure enterocin L50 was also the most effective bacteriocin against the

foodborne pathogens Cl. perfringens and Cl. botulinum (MICs of about 475 ng ml−1). Although crude pediocin PA-1 and nisin A were very active against clostridia, high concentrations of pure bacteriocins (MICs from 648 to 1575 ng ml−1) were required to inhibit these micro-organisms. Pediocin PA-1 and enterocin L50 were the most efficient bacteriocins against Listeria monocytogenes, with MICs between 30 and 187 ng ml−1 and between 151 and 475 ng ml−1, respectively. S. aureus strains were weakly inhibited by lactocin S and nisin A, while pediocin PA-1 inhibited growth of only S. aureus FRI137. Nevertheless, enterocin L50 was able to inhibit growth of the five pathogenic S. aureus strains tested, at concentrations from 163 to 740 ng ml−1.

Discussion In recent years, several reports dealing with bacteriocins produced by LAB have been published (see Introduction). However, studies on bacteriocin performance under identical conditions have been given little attention. From our collection of bacteriocinogenic LAB, all isolated from Spanish dry-fermented sausages, four different bacteriocin-producing strains were selected. The bacteriocins were purified to homogeneity by modifying the procedure described by Nissen-Meyer et al. (1992). This purification procedure allowed high and reproducible yields of both lantibiotics (nisin A and lactocin S) and class II bacteriocins (pediocin PA-1 and enterocin L50). An increase in nisin activity was observed after the last chromatographic step, which may be due to the removal of inhibitors of bacteriocin activity during the purification and/or to a conformational change of the mol-

Table 2. Parameters from the purification of enterocin L50, pediocin PA-1, nisin A and lactocin S Bacteriocin producer Enterococcus faecium L50 Pediococcus acidilactici Z102 Lactococcus lactis BB24 Lactobacillus sake V18 a b

Increase in specific activitya

Yield (%)

Protein (µg)b

1·1×105 6·0×104 9·0×106 1·6×105

80 44 23 000 26

208 103 126 21

Bacteriocin units (BU) per millilitre divided by optical density at 254 nm. Amount of pure bacteriocin obtained from 1-litre cultures of the bacteriocinogenic strains.

Comparative activity of bacteriocins 295

ecule to a more active form in the hydrophobic solvent. This increase in biological activity has also been reported for some pediocin-like bacteriocins, such as pediocin PA-1 (Henderson et al. 1992), enterocin A (Aymerich et al. 1996), enterocin P (Cintas et al. 1997), curvacin A (sakacin A) (Tichaczek et al. 1992, Holck et al. 1992), and sakacin P (Tichaczek et al. 1992). The purification processes resulted in sufficient quantities of

pure and stable enterocin L50, pediocin PA-1, nisin A and lactocin S to determine their MICs against a wide variety of Gram-positive bacteria, including spoilage and foodborne pathogenic bacteria. The antimicrobial spectra of purified bacteriocins resembled that of culture supernatants, which suggests that the microbial inhibition found in the crude extracts was due to these bacteriocins and not to other bacteriocins or compounds. In

Table 3. MIC of enterocin L50, pediocin PA-1, nisin A and lactocin S against selected indicator microorganisms Indicator species

Lactobacillus acidophilus bulgaricus casei curvatus fermentum helveticus sake salivarius Lactococcus cremoris lactis lactis Leuconostoc cremoris Pediococcus pentosaceus pentosaceus pentosaceus Clostridium sporogenes tyrobutyricum tyrobutyricum Enterococcus faecalis Listeria innocua Propionibacterium sp. Propionibacterium sp. Prop. acidipropionici Staphylococcus carnosus Bacillus cereus Clostridium perfringens botulinum Listeria monocytogenes monocytogenes monocytogenes monocytogenes monocytogenes Staphylococcus aureus aureus aureus aureus aureus a

Strain

4356 11842 334 2739 9338 15009 2714 2747 CNRZ117 CNRZ148 CNRZ150 DB1275 FBB61 FBB63 PC1 C22/10 3,5CT 1754 EF BL86/26 P4 P6 573 MC1 9139 376 551 7973 LI5sv1/2 5105 LI1sv4 ScottA 137 196E 349 361 472

Sourceb

ATCC ATCC ATCC NCFB ATCC ATCC NCFB NCFB INRA INRA INRA TNO TNO TNO TNO TNO TNO NCDO TNO TNO TNO TNO NCDO TNO ATCC CECT CECT NCTC FVM NCTC FVM FVM FRI FRI FRI FRI FRI

Purified bacteriocin MIC (ng ml−1)a Enterocin L50

Pediocin PA-1

Nisin A

Lactocin S

13 60 13 114 260 57 77 NID 103 100 59 109 7 120 107 475 443 459 193 457 189 218 213 225 187 475 471 278 475 377 151 463 740 163 404 377 470

NID 6 2888 7 121 3 9 2389 NID NID NID NID 0·7 10 NID 623 NID NID 133 131 0·2 0·2 0·2 126 3086 1113 648 160 100 187 93 30 305 NID NID NID NID

44 8 4 12 121 3 13 12 25 1544 1443 41 2 12 27 690 1506 795 812 852 255 398 377 776 778 1515 1575 762 873 434 772 681 686 633 762 817 781

285 125 187 38 360 85 93 106 100 NID NID 123 38 91 75 765 660 533 425 485 328 316 315 388 1990 800 862 785 725 740 650 636 1116 660 890 770 1200

Bacteriocin concentration inhibiting growth of indicator micro-organisms by 50%; NID, no inhibition detected at 3·75 µg enterocin L50 ml−1 or 6·0 µg pediocin PA-1, nisin A or lactocin S ml−1. Two replicates of each MIC assay were performed. b Abbreviations are the same as for Table 1.

296 L. M. Cintas et al.

this context, it should be noted that more than one bacteriocin may be produced by a single bacterial strain (Nes et al. 1996, Casaus et al. 1997). Interestingly, crude pediocin PA-1 and nisin A were very active against clostridia, but high concentrations of pure bacteriocins were required to inhibit these micro-organisms, which may indicate that the inhibition produced by the supernatants was due to the combined action of the bacteriocins and other compound(s) that were removed during the purification. The four bacteriocins showed a broad antimicrobial spectrum and were active against most of the LAB tested, but pediocin PA-1 did not inhibit the growth of the three Lactococcus lactis Subsp. strains tested, The resistance of lactococci to pediocin-like bacteriocins, such as sakacin A, sakacin P and enterocin A has been previously reported by Aymerich et al. (1996). Interestingly, enterocin L50 inhibited growth of lactococci, which led us to speculate that its mode of action and the bacteriocin receptors, if any, may be different to that of pediocin-like bacteriocins. Enterocin L50 and pediocin PA-1 have a strong antimicrobial activity against Listeria monocytogenes, a reluctant micro-organism which frequently causes foodborne listeriosis (Farber and Peterkin 1991, Lepoutre et al. 1992). However, nisin A is not very effective against L. monocytogenes, a finding that has been already reported by other authors (Benkerroum and Sandine 1988, de Vos et al. 1993). Despite that, nisin A is generally accepted as a food preservative in several countries, particularly for the inhibition of growth of Clostridium spp. in cheese and canned foods (Hurst 1981, Delves-Broughton 1990), but nisin A fails to work as a preservative agent in meat and meat products (Delves-Broughton 1990). Explanations for this altered effectiveness of nisin A include its binding to meat particles and surfaces, uneven distribution, poor stability, sensitivity to meat enzymes and interference with phospholipid emulsifiers (Delves-Broughton 1990). Interestingly, B. cereus ATCC9139, a micro-organism known to cause two different foodborne illnesses (Doyle 1988), was sensitive to pure enterocin L50 and nisin A, but

not to the crude bacteriocin extracts. This phenomenon may be due to the higher bacteriocin amounts used in the microtitre plate assays and/or to an increased sensitivity of B. cereus as the result of growth conditions in liquid medium. Enterocin L50 also displayed antimicrobial activity against Cl. botulinum and S. aureus, two pathogenic bacteria which must be controlled in food as they are frequently associated with food poisoning (CDC 1979, Smith and Palumbo 1981, Frazier and Westhoff 1985). Since the four bacteriocin-producing LAB studied in this work have been isolated from meat products, it is tempting to speculate that they will be highly competitive in meat systems and able to produce bacteriocins in situ, and consequently be more suitable as food-preserving micro-organisms than microorganisms isolated from different environments and producing the same bacteriocins. From our results, it is interesting to note that enterocin L50 and pediocin PA-1 complement each other, especially in their antimicrobial activity against L. monocytogenes and S. aureus. Since these bacteriocins have different antimicrobial spectra they may have different modes of action, and for that reason each of them may be active against the bacteria resistant to the other bacteriocin(s). In conclusion, the use of either a mixture of these bacteriocins or genetically engineered bacteria producing multiple bacteriocins may be a useful approach to reduce the frequency at which resistant populations develop, and to improve the hygienic quality and to extend the shelf-life of meat and meat products.

Acknowledgements The authors thank I. F. Nes (Laboratory of Microbial Gene Technology, A° s, Norway) for critical reading of the manuscript and helpful discussions. The authors acknowledge that some of the results presented in this paper had been pre´ viously published (Cintas et al. 1995, Rodrıguez et al. 1995, 1997). This work was partially supported by ´ grant ALI91-0255 from the Comisio ´ n Interministerial de Ciencia y Tecnologıa (CICYT),

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Spain, and the Commission of European Communities (Contract Biot-CT91-0263). Luis M. Cintas and Pilar Casaus were recipients of grants within the National Plan for Training Researchers (F. P. I.) from the Min´ isterio de Educacion y Ciencia, Spain.

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