Characterization of Enterocin 1146, a Bacteriocin from Enterococcus Faecium Inhibitory to Listeria Monocytogenes

Characterization of Enterocin 1146, a Bacteriocin from Enterococcus faecium Inhibitory to Listeria monocytogenes

EUGENIO PARENTE1 and COLIN HILL*

The National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, The Irish Republic Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021

(Received for publication November 12, 1991)

ABSTRACT cultures has been proposed for Cheddar cheese (37), Fontina cheese (6), and water-buffalo Mozzarella cheese (57). Enterococcus faecium DPC 1146 produces a bacteriocin, In this report we describe the characterization of enterocin 1146, which is inhibitory to Listeria monocytogenes. Enterocin 1146 was produced in GM17 and in milk. The bacterio enterocin 1146, a bacteriocin produced by E. faecium cin was partially purified by ammonium sulfate precipitation. Its DPC1146, which is relatively specific to Listeria spp. molecular weight, estimated by SDS-PAGE, was 3.0 kDa. It could be stored at -20°C without loss of activity, but pH had a marked MATERIALS AND METHODS effect on enterocin 1146, which was more stable at both high (up to 120°C) and low temperatures (4°C) at pH 5 than at pH 7 and Strains and media 9. The sensitivity of 57 strains belonging to 35 different species Enterocin 1146 is produced by E. faecium DPC1146. Unless was studied using a critical dilution assay. L. monocytogenes and otherwise noted L. innocua DPC 1770 was used as the indicator L. innocua were most sensitive; enterocin 1146 had a bactericidal strain. A complete list of the strains, their growth media, and effect on Listeria. Starter and nonstarter lactic acid bacteria temperature of incubation used in this study is shown in Table 1. (except Lactobacillus sake) were insensitive or relatively resistant All strains were maintained frozen at -80°C in 25% glycerol. to the bacteriocin. Genetic determinants for bacteriocin production Enterococci were routinely propagated in GM17 (M17, Difco and immunity do not appear to be plasmid borne. Laboratories, Detroit, MI + 0.5% glucose). GM17 dialysate (DGM17) was prepared by dissolving M17 in water with the appropriate amount of glucose at 10X concentration. The solution was then dialyzed against sterile distilled water and the permeate Bacteriocins are bacterial proteins which are usually, was sterilized and used as a growth medium. but not always, inhibitory to species and strains closely related to the producer. Several bacteriocins produced by Measurement of bacteriocin activity lactic acid bacteria have been characterized (27). While A critical dilution assay was used. Culture supernatant or most have a narrow inhibitory spectrum, some, like pediocin filter sterilized bacteriocin solutions were serially diluted (two A (77), pediocin PA-1 (32), pediocin AcH (8), sakacin A fold) in GM17 broth, and 10 ul aliquots were spotted on GM17 (34), are also active against foodborne pathogens, like plates and dried for 30 min. The plates were overlaid with 3 ml (7 ml for Clostridia) of the appropriate soft (0.7% agar) medium, Listeria monocytogenes, and thus have a potential as natu preinoculated with 0.1 ml of an overnight culture of the indicator, ral food preservatives. and incubated at the appropriate temperature. One arbitrary unit The ability of enterococci to produce bacteriocins, (AU) was defined as the reciprocal of the highest dilution of some of which display a broad inhibitory spectrum, is well enterocin 1146 giving a zone of inhibition on the indicator lawn known (9). Enterocin E1A, a well-characterized bacteriocin (32). When a clear inhibition zone was followed by a turbid one, from Enterococcus faecium (22,23), has been shown to be the critical dilution was taken to be the average of the final two active against Listeria (3). According to a number of recent dilutions. reports (2,26), the ability to inhibit L. monocytogenes may be relatively widespread among enterococci. Enterococci Bacterial counts Viable bacterial cells were enumerated using a Spiral Plater have caused foodborne infections only in very rare in (Spiral Systems, Inc., Cincinnati, OH). E. faecium DPC1146 was stances and evidence on their role as foodborne pathogens enumerated on GM17 agar after incubation for 24 h at 37°C. L. are scant or lacking (20). They can be isolated from innocua DPC 1770 was enumerated on trypticase soy agar (BBL artisanal cheeses and starters (70), and their use in starter Microbiology Systems, Cockeysville, MD) + 0.6% yeast extract (Oxoid, Ltd., Basingstoke, Hampshire, England) after incubation for 48 h at 30°C. Colonies were enumerated using a Protos ' Present address: Dipartimento di Biologia, Difesa e Biotecnologie Colony Counter (Ai Cambridge, Ltd; Pannisford, Cambridge, Agro-Forestali, Universita' della Basilicata, 85100 Potenia Italy. England).

JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 498 PARENTE AND HILL Table 1. Bacterial strains, media, and temperature of incubation used in this study. Relative sensitivity of a strain to partially purified enterocin 1146 (ppE1146) is calculated as the ratio between the activity of the bacteriocin (AU/ml) using that strain as indicator to that obtained on L. innocua DPC 1770 (76800 AU/ml).

Strain Medium1 Temp. (°C) Source2 Relative sensitivity

Bacillus cereus ATCC9139 TSB 37 TNO 0 B. subtilis BD630 TSB 37 RuG 0 B. subtilis OG1 TSB 37 RuG 0 Clostridium butyricum NCD01713 RCM 30 DPC 0.083 C. sporogenes NCD01789 RCM 37 DPC 0 C. sporogenes C 22/10 RCM 37 TNO 0 C. perfringens NCDO1800 RCM 37 DPC 0.032 C. tyrobutyricum NCD01754 RCM + SL 30 TNO 0 C. tyrobutyricum NCD01756 RCM + SL 30 DPC 0 C. tyrobutyricum NCDO1790 RCM + SL 30 DPC 0 C. tyrobutyricum 3.5 RCM + SL 30 TNO 0 Enterococcus faecium DPC 11463 GM17 37 DPC 0 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021 E. faecium DPC3342" GM17 37 DPC 0.004 E. faecalis NCD0581 GM17 37 DPC 0.063 E. faecalis NCDO610 GM17 37 DPC 0.063 £./aeca/isDPC1142 GM17 37 DPC 0.063 E. faecalis 1 GM17 37 TNO 0.042 Lactobacillus acidophilus ATCC4356 MRS 37 TNO 0 L. casei ATCC334 MRS 37 TNO 0 L. casei ssp. pseudoplantarum DPC2136 MRS 37 DPC 0 L. curvatus NCFB2739 MRS 30 TNO 0.001 L. delbrueckii subsp. bulgaricus ATCC1184 MRS 42 TNO 0.002 L. delbrueckii subp. lactis DPC 1125 MRS 37 DPC 0 L. fermentum ATCC9338 MRS 37 TNO 0 L. helveticus ATCC15009 MRS 42 TNO 0 L. helveticus DPC 1130 MRS 42 DPC 0.005 L. plantarum NCD01193 MRS 37 TNO 0.001 L. reuteri DSM20016 MRS 37 TNO 0 L. sake NCFB2714 MRS 30 TNO 0.500 L. salivarius NCFB2747 MRS 37 TNO 0 Lactococcus lactis subsp. lactis MG1614 GM17 30 DPC 0.063 L. lactis subsp. lactis DPC2612 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DPC938 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DPC979 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DPC990 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DRC3 GM17 30 DPC n.t.5 L. lactis subsp. cremoris DPC2645 GM17 30 DPC 0 L. lactis subsp. cremoris CNRZ117 GM17 30 TNO 0 Leuconostoc cremoris DB1275 MRS 25 TNO 0 Leuconostoc subsp. DPC225 MRS 25 TNO 0 Leuconostoc subsp. DPC 1061 MRS 25 DPC 0 Listeria innocua DPC 1770 TSB YE 30 DPC 1 L. innocua BL86/26 TSB YE 30 TNO 0.667 L. monocytogenes 9 TSB YE 30 DPC 0.500 L. monocytogenes DPC 1771 TSBYE 30 DPC 0.667 L. monocytogenes NCTC5348 TSB YE 30 DPC 4.000 L. monocytogenes Scott A TSBYE 30 DPC 1 Pediococcus pentosaceus FBB63 MRS 30 TNO 0.083 P. pentosaceus PCI MRS 30 TNO 0 P. acidilactici NCFB2767 MRS 30 DPC 0.002 Propionibacterium acidipropionici NCFB563 YGL 30 Cranfield 0 Propionibacterium spp. P4 YGL 30 Cranfield 0 Propionibacterium spp. P6 YGL 30 Cranfield 0 Salmonella typhimurium ATCC 14028 TSB 37 DPC 0 Staphylococcus aureus DPC 1772 TSB 37 DPC 0 S. carnosus MCI TSB 37 TNO 0 Streptococcus thermophilus DPC 1780 GM17 37 DPC 0 S. thermophilus DPC2227 GM17 37 DPC 0 RCM = Reinforced clostridial medium (Oxoid Ltd.); RCM + SL = RCM + 0.5% sodium lactate (BDH Chemicals Ltd.); TSB = trypticase soy broth (BBL Microbiology Systems); TSBYE = TSB + 0.6% yeast extract (Oxoid); GM17 = M17 (Difco Laboratories) + 0.5% dextrose (Oxoid); MRS from Difco; YGL = Yeast Glucose Lemco Broth (29). Top and bottom media for the critical dilution assay used for the measurement of sensitivity were obtained adding, respectively, 0.7% and 1.5% agar bacteriological to the broth media listed. TNO: Dr. B. ten Brink, TNO Nutrition and Food Research, Zeist, The Netherlands. RuG: Prof. G. Venema, University of Groningen, The Netherlands. DPC: National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork. Cranfield: Cranfield Institute of Technology, Cranfield Bedford, England. Producer of enterocin 1146. Bac- derivative of DPC 1146. Not tested.

JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 BACTERIOCIN FROM ENTEROCOCCUS FAECIUM 499 Production of enterocin 1146 in different media Adsorption of enterocin 1146 to sensitive and resistant cells An overnight culture of E. faecium DPCl 146 was used to Cells from an overnight culture of L. innocua DPCl770 were inoculate (105 CFU/ml) GM17 broth, 10% skim milk (Oxoid), and collected by centrifugation, washed twice in 50 mM potassium Elliker lactic broth (12). The inoculated media were incubated at phosphate buffer, pH 7, and then diluted 1:10 in the same buffer 37°C, and samples were removed for the measurement of bacte (obtaining 2.7 x 10s CFU/ml). Controls were buffer alone or cell riocin activity and viable cell counts. suspensions of E. faecium DPCl 146 (6.1 x 107 CFU/ml) or its Bac- derivative, DPC3342 (6.8 x 107 CFU/ml), obtained as de Preliminary purification of enterocin 1146 scribed above. Partially purified enterocin 1146 was added to the Bacteriocin was precipitated from GM17 culture supernatant control and cell suspensions to a final concentration of 300 AU/ for 1 h in the presence of 55% (NH4)2S04 (Merck, Darmstadt, ml. The samples were immediately vortexed and incubated at Germany) at 4°C. The precipitate was resuspended in sterile 0.05 30°C. Tubes were removed at five sampling times (5 to 90 min) M potassium phosphate buffer, pH 5.5, and dialyzed (Visking and centrifuged for 3 min. The supernatant was separated from the VT10 dialysis tubing, Medicell, International, Ltd; London) over cells and assayed for bacteriocin activity by the critical dilution night at 4°C against the same buffer. The bacteriocin solution was assay. Cell suspensions of the resistant strains (DPCl 146 and then filter sterilized through a Millipore HV filter and stored DPC3342) were assayed for residual bacteriocin activity only at frozen at -20°C. This bacteriocin preparation was designated 15 and 30 min. partially purified enterocin 1146 (ppE1146) and used in all the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021 following experiments. The activity of the partially purified prepa Effect of enterocin 1146 on resting cells of L. innocua DPC1770 ration, assayed using Listeria innocua DPCl770 as an indicator, Cells from an overnight culture of L. innocua DPCl770 were was 76,800 AU/ml and remained constant during 4 months of washed and resuspended in sterile 50 mM potassium phosphate storage at -20°C. buffer, pH 7, to obtain about 108 CFU/ml; ppE1146 was then added to a final concentration of 0, 75, or 1200 AU/ml and plate Effect of enzymes counts were carried out after 10, 30, 60, 120, 180, min at 30°C. Enzymes, including trypsin (type III-S, Sigma Chemical Co., Poole, Dorset, England), a-chymotrypsin (type II, Sigma), protei nase k (Sigma), pronase E (type XIV, Sigma), pepsin (BDH, RESULTS AND DISCUSSION Chemicals Ltd; Poole, England) and catalase (Sigma) were dis solved in 0.05 M Tris pH 7.5, 0.005 M CaCl, (except for pepsin Enterococcus faecium DPCl 146 was identified as a which was dissolved in 0.02 N HC1) to obtain a final concentra bacteriocin producer during a screening of the National tion of 1 mg/ml; partially purified enterocin 1146 was added to Dairy Products Research Centre collection. Of 224 strains obtain about 3200 AU/ml and the mixtures were incubated at (belonging to the genera Enterococcus, 'Lactobacillus, 37°C for 1 h. At the end of the incubation the residual activity of Lactococcus, Pediococcus, and Leuconostoc), it was the the bacteriocin was assayed as described above. Controls included the bacteriocin in buffer without the enzymes, the enzymes alone, only one to show inhibitory activity against L. innocua and and the buffers alone. L. monocytogenes. The inhibitory substance was nondialyz- able, insensitive to catalase, and sensitive to a number of Effect of pH and temperature on stability proteolytic enzymes (pronase E, pepsin, trypsin, a-chymot The heat resistance of the bacteriocin at pH 5, 7, or 9 was rypsin, and proteinase K). The inhibitory activity of E. tested by exposing the bacteriocin solutions to various tempera faecium DPCl 146 therefore complied with a number of the ture-time combinations and measuring the bacteriocin activity criteria for bacteriocins (36) and was designated enterocin after rapid cooling and adjustment to pH 7. To investigate if the 1146. inactivation of enterocin 1146 at neutral or alkaline pH was Growth of E. faecium DPCl 146 and production of irreversible, samples of bacteriocin were also adjusted to pH 7 and 9, incubated at 37°C for 2 h, and adjusted to pH 3 or 5; the enterocin 1146 were studied in GM17, Elliker lactic broth, incubation was then continued for 4 h and activity was assayed and reconstituted skim milk. The results are shown in Fig. every 2 h. 1. Highest cell numbers and bacteriocin activity were ob tained in GM17. In GM17, enterocin 1146 is produced Estimation of molecular weight early, peaking during late log phase. This is not uncommon, Swank and Munkres (35) urea-SDS-polyacrylamide gels were and it has been proposed that the subsequent reduction in loaded with ppE1146. Controls included ammonium sulfate pre activity may be due to poor stability of the bacteriocin and/ cipitate from a DGM17 culture of DPC3342, a Bac- derivative of or to proteolytic enzymes produced by the culture (5,28). DPCl 146 (see below), and trypsin treated ppE1146. Polypeptide Growth and bacteriocin production in milk were slow, but Molecular Weight Calibration Kit (Pharmacia AB, Uppsala, Swe 1600 AU/ml were obtained after 24 h. These results are in den) was used as a standard. After electrophoresis at 2 mA/cm for 14 h, the gels were divided and half was treated according to contrast with those of Geis et al. (14) who found that Bhunia et al. (7) for detection of bacteriocin activity, while half production of different bacteriocins by lactococci was higher was stained according to Giulian et al. (15). in Elliker lactic broth than in Ml 7. Proteins and peptides present in a rich medium like GM17 make the purification DNA isolation and plasmid curing of bacteriocins more complicated. A substrate obtained by Plasmid DNA was isolated using the method of Anderson eliminating large molecular weight components from a and McKay (1). Plasmids were separated on 0.65% horizontal complex medium was used for the production of pediocin agarose gels and sized using Lactococcus lactis subsp. lactis AcH (8). Therefore, growth and production of enterocin biovar diacetylactis DRC3 plasmids as molecular weight stan 1146 were tested in GM17 dialysate (DGM17); although dards (27). Curing was carried out by prolonged growth in GM17 + 20 |J.g/ml acridine orange at high temperature (48°C). Pronase this medium supported good growth of the producing strain, E (Sigma) was added at 0.5 |J.g/ml to inactivate enterocin 1146 a maximum bacteriocin activity of only 400 AU/ml was and improve the probability of recovery of Bac- derivatives. obtained after 6 h.

JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 500 PARENTE AND HILL A partially purified bacteriocin preparation (ppEl 146) Table 2. Effect of pH and temperature on the stability of enterocin was obtained as described in the methods. The effect of pH 1146 (1200 AU/ml). and temperature on the stability of ppE1146 was investi gated (Fig. 2). Heating at temperatures higher than 60°C Temp. (°C) Time AU/ml at pH resulted in partial or total inactivation. Partially purified (h) 5 7 9 enterocin 1146 is relatively unstable at temperatures greater than -20°C (Table 2). The pH of bacteriocin solutions had 4 8 1200 600 400 a dramatic effect on their stability; even at 4°C losses of 24 800 400 200 activity at neutral and alkaline pH were more intense than at pH 5. The inactivation at neutral and alkaline pH was 21 2 1200 600 400 partially reversible. If a bacteriocin solution at pH 7 or 9 8 1200 600 300 was incubated at 37°C, the activity lost after 2 h of 24 600 150 200 incubation could be restored by adjusting the pH to 3 but not to 5 (not shown). At acid pH the bacteriocin is remark 37 2 1200 600 150 ably heat stable although some loss of activity is observed 8 300 100 100 at temperatures higher than 80°C. Even at 120°C for 10 24 300 100 50 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021 min, 33% of the activity was retained at pH 5 while no activity was left at pH 7 and 9. Enterocin 1146 stability is The molecular weight of enterocin 1146 was estimated less than nisin (18) and pediocin AcH (8) but comparable to be 3000 Da by direct detection of antimicrobial activity with pediocin PA-1 (16). after electrophoresis on SDS-PA gels (Fig. 3). The molecu 10 • lar weight of enterocin E1A, a bacteriocin produced by E. faecium El A, was estimated to be 10,000 Da by gel filtra tion (22). Bacteriocins produced by lactic acid bacteria have molecular weights ranging from 2700 (8) to more than 15,000 Da, although complexes of molecular weight ex ceeding 200 kDa are sometimes found (21). 12 3 4

17201

1 4632

time (h) 8235 Figure 1. Growth of E. faecium DPC1146 and production of enterocin 1146 in different media. O GM17; A Elliker lactic 63 83 broth; • reconstituted skim milk. Empty symbols: log (CFUIml); closed symbols: bacteriocin activity (1000 x AU/ml). TbUU - 3000

3 2556 < 1200 - Jr \ Figure 3. Estimation of molecular weight of enterocin 1146 by SDS-PAGE. Lane 1 trypsin inactivated partially purified enterocin 800 - 1146 (ppE1146.), lane 2 ppE1146, lane 3 ammonium sulfate \ K precipitate from culture of DPC3342 (Bac-) in M17 dialysate (DGM17), lane 4 ppEl 146 from DGM17. Molecular weight stan dards are also indicated. 400 - k.. i-A. \ • •m A k The plasmid profile of E. faecium DPC1146 and de rivatives obtained after acridine orange/high temperature . fcm\ curing is shown in Fig. 4. DPC1146 has a single detectable

0 - • i r | l | i | i | i 'ta-r—i plasmid of 55 MDa. DPC3342, a nonproducing (Bac-), 0 20 40 60 80 100 120 140 sensitive (Imm-), derivative obtained after acridine orange mutagenesis retained the plasmid while three producing T (C) (Bac+), insensitive (Imm+) derivatives (DPC3347, DPC3348, Figure 2. Effect of heating at 60 to 120°C for 10 min on the stability DPC3349) are plasmid free. DPC3342 was the only Bac- of enterocin 1146 at different pH. • pH 5; • pH 7; • pH 9. derivative isolated during repeated curing experiments in

JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 BACTERIOCIN FROM ENTEROCOCCUS FAECIUM 501 which several thousand colonies were examined for bacte- it is similar to pediocin PA-1 (16) which, however, is more riocin production. In contrast, the frequency of isolation of active against Pediococci and Lactobacilli than against plasmid-free derivatives was higher than 50% after pro Listeria (32). The antimicrobial spectrum of enterocin 1146 longed curing. Bacteriocin production and immunity are is similar to that of enterocin E1A (22). L. innocua DPC1770 usually genetically linked. In contrast, the sensitivity of was chosen as an indicator for any further experiment DPC3342 to ppE1146 was only slightly higher than the because, while being nonpathogenic, its sensitivity to parent strain. We conclude that the determinants for enterocin ppE1146 is comparable to that of L. monocytogenes. 1146 production and immunity are not located on the The kinetics of adsorption of enterocin 1146 to sensi plasmid. They might be located on the chromosome or on tive and resistant cells is shown in Fig. 5. Adsorption of a highly stable plasmid which could not be detected by enterocin 1146 is aspecific and rapid (5 min in L. innocua vertical or horizontal agarose gel electrophoresis even after DPC1770). In this respect enterocin 1146 is similar to most prolonged runs. All other E. faecium bacteriocin for which bacteriocins of lactic acid bacteria (21). data are available appear to be plasmid encoded (24,33). 400 -i However, chromosomally encoded bacteriocins are not

uncommon among other lactic acid bacteria (4,19). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021 # ^ # ^ .f 300 I hO-O-O

200 - 3 <

100 -

time (min) Figure 5. Adsorption of enterocin 1146 by sensitive (L. innocua DPC1770, 2.7 x10s CFUIml) and resistant (E. faecium DPC1146, producer strain, 6.1 x 107 CFUIml, and DPC3342, Bac- deriva tive, 6.8 x 107 CFUIml) cells in 50 mM potassium phosphate buffer, pH 7. O control (no cells); • L. innocua DPC1770; A E. faecium DPC1146; • E. faecium DPC3342.

Enterocin 1146 had a bactericidal effect on L. innocua suspended in potassium phosphate buffer; viable cell num bers rapidly decreased during the first 30 min but remained stable thereafter (Fig. 6). The kinetics of death of indicator Figure 4. Plasmid profile ofE. faecium DPC1146 and its deriva cells treated with enterocin 1146 is similar to that observed tives. Lane 1: L. lactis subsp. lactis biovar diacetylactis DRC3, for lactacin B (4) and a bacteriocin produced by Leuconostoc used as molecular weight standard (molecular weights from top to gelidum (17). Other bacteriocins, like sakacin A (34), have bottom, in Mda; 52, 40, 34, chromosome, 5.5); lane 2: DPC1146, a slower but more prolonged bactericidal effect. parent strain; lane 3; DPC3342 Bac- derivative; lanes 3 to 5: The potential of bacteriocins produced by lactic acid DPC3347, DPC3348, DPC3349, Bac+ lmm* plasmidless derivatives. bacteria as novel food preservatives is well recognized (21). Unfortunately, nisin and most bacteriocins with a The relative sensitivity of 57 strains of lactic acid relatively large inhibitory spectrum affect starter and bacteria, spoilage organisms, and foodborne pathogens to nonstarter lactic acid bacteria, which is not desirable in enterocin 1146 is shown in Table 1. All Listeria strains cheese and other fermented foods. The relative insensitivity tested, including the known human pathogens L. monocyto of most lactic acid bacteria to enterocin 1146 compared genes Scott A (13) and NCTC5348, were highly sensitive. with the high sensitivity of Listeria spp. are relatively Lactic acid bacteria, with the exception of a single Lacto unique and promising properties of this bacteriocin. While bacillus sake strain (relative sensitivity, RS = 0.5), were several other bacteriocins have been shown to be active relatively insensitive (the plasmid-free Lactococcus lactis against L. monocytogenes (25), studies on the their effect subsp. lactis MG1614, enterococci, and P. pentosaceus on Listeria are relatively rare (17,32). The effect of enterocin FBB63, with RS between 0.04 and 0.08) or resistant (RS 1146 on the growth and survival of Listeria in buffer, broth, less than 0.005) to the bacteriocin. High doses of ppEl 146 and milk is reported in Parente and Hill (30). were inhibitory to Clostridium butyricum and C. perfringens. All the other foodborne pathogens were insensitive. ACKNOWLEDGMENTS These properties could make enterocin 1146 particularly useful in food systems where fermentations must proceed E. Parente's stay in Moorepark was financed by a scholarship from freely while Listeria spp. must be inhibited. In this respect Consiglio Nazionale delle Ricerche, Rome, Italy.

JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 502 PARENTE AND HILL

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JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992