Bacteriocin-Mediated Inhibition of Clostridium botulinum Spores by Lactic Acid Bacteria at Refrigeration and Abuse Temperaturest

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1991, p /91/ $02.00/0 Copyright 1991, American Society for Microbiology Vol. 57, No. 12 Bacteriocin-Mediated Inhibition of Clostridium botulinum Spores by Lactic Acid Bacteria at Refrigeration and Abuse Temperaturest AMECHI OKEREKE AND THOMAS J. MONTVILLE* Department of Food Science, New Jersey State Agricultural Experiment Station, Rutgers, The State University of New Jersey, New Brunswick, New Jersey Received 8 July 1991/Accepted 12 September 1991 The bacteriocinogenicity of Lactococcus lactis ATCC 11454, Pediococcus pentosaceus ATCC 43200, P. pentosaceus ATCC 43201, Lactobacilus plantarum BN, L. plantarum LB592, L. plantarum LB75, and Lactobacillus acidophilus N2 against Clostridium botulinum spores at 4, 10, 15, and 35 C was investigated by modified deferred and simultaneous antagonism methods. AU the strains, except L. acidophilus N2, produced inhibition zones on lawns of C. botulinum spores at 30 C. L. plantarum BN, L. lactis ATCC 11454, and P. pentosaceus ATCC and were bacteriocinogenic at 4, 10, and 15 C. Supplementation of brain heart infusion agar with 0 to 5% NaCl increased the radii of inhibition zones during simultaneous antagonism assays. Detectable bacteriocin activities were extracted from freeze-thawed agar cultures of L. plantarum BN and L. lactis ATCC which had been grown at 4 and 10 C. These results suggest that low levels of L. plantarum BN or L. lactis ATCC 11454, in the presence of 3 or 4% NaCl, could be formulated into minimally processed refrigerated food products for protection against possible botulism hazards. Some strains of the lactic acid bacteria produce bacteriocins (8, 11, 28) that are inhibitory to Listeria monocytogenes (9, 23), Staphylococcus spp. and Clostridium perfringens (4, 12, 17), proteolytic and nonproteolytic Clostridium botulinum types A and B spores (19), and a host of other gram-positive and gram-negative nonpathogenic bacteria (7). Although C. botulinum spores occur naturally in soil (25), they occasionally contaminate raw meats (21) intended for the manufacture of minimally processed foods. Because of their relatively high resistance to heat, low water activity (aj), and high salt, they survive the curing and pasteurization treatments (10) given to minimally processed meat products. Proteolytic strains of C. botulinum grow optimally at 37 C and slowly at 10 C (2, 24). Nonproteolytic strains grow and produce toxins at temperatures as low as 3.3 to 4.0 C (14, 26). It is highly probable that storage of minimally processed refrigerated foods at temperatures higher than 10 C will result in botulism hazard if spores survived the pasteurization process (18). Sodium chloride and nitrite, at refrigeration temperatures (20), synergistically inhibit the outgrowth of C. botulinum spores in cured meats. The implication of nitrite in nitrosamine formation in cured meats led to the search for an alternative antibotulinal agent. Because bacteriocins are produced by naturally occurring foodborne organisms (11) and inhibit many gram-positive pathogens (28), they might be useful antibotulinal agents in nonfermented cured meats. Bacteriocins can be used to inhibit foodborne pathogens by formulation of viable cells of a bacteriocinogenic strain into the food, by addition of bacteriocin contained in a fermentation by-product such as whey, or by direct addition of purified bacteriocin (3). Applications based on growth of the lactic cells would require that they be added at subspoilage levels and that the cells be able to grow and produce bacteriocin at both refrigeration and abuse temperatures. * Corresponding author. t This is paper D of the New Jersey State Agricultural Experiment Station Inocula as low as 105 CFU/ml produce inhibition zones on lawns prepared with 104 CFU of C. botulinum spores per ml (19). Bacteriocin activity in food is highly dependent on the properties of the given food system (3). It is generally accepted that nisin is most effective at a ph of <6.0 in low-fat and -protein foods. We demonstrated (19) antibotulinal activities by subspoilage levels of seven bacteriocinogenic strains of lactic acid bacteria. In the present study we investigated the ability of these strains to grow, produce bacteriocins, and inhibit C. botulinum spores at both refrigeration and abuse temperatures in the presence of various concentrations of sodium chloride which might be found in cured meats. The activities of different inoculum levels of the bacteriocinogenic strains against lawns prepared with different concentrations of C. botulinum spores were also investigated. In this communication we present the first evidence that bacteriocinogenic lactic acid bacteria produce bacteriocins at refrigeration temperatures. The paper also describes the effect of sodium chloride on bacteriocinogenicity. MATERIALS AND METHODS Cultures and media. The maintenance and propagation of all the bacteriocin-producing strains used in this study were as previously described (19). Frozen stock cultures were maintained at -80 C in Lactobacillus MRS broth (Difco Laboratories, Detroit, Mich.) plus 20% glycerol. Lactococcus lactis ATCC 11454, Lactobacillus acidophilus N2, Lactobacillus plantarum LB75, L. plantarum LB592, L. plantarum BN, Pediococcus pentosaceus ATCC (also designated FBB61 [4] prior to its deposition with the American Type Culture Collection), and P. pentosaceus ATCC were used as the bacteriocin-producing strains. L. plantarum ATCC 8014, a strain that does not produce bacteriocins, was used as a negative control. Unless otherwise stated, all bacteriocin-producing cultures were grown overnight at 30 C in MRS broth. Plate counts of standardized overnight cultures were on MRS broth solidified with 1.5% Bacto Agar (Difco). Bacteriocin production and detection

2 3424 OKEREKE AND MONTVILLE were on brain heart infusion (BHI) broth (Difco) supplemented with filter-sterilized catalase (Sigma Chemical Co., St. Louis, Mo.) at a level of 300 IU/ml to eliminate possible inhibition due to H202 (6) and solidified with either 0.7 or 1.5% agar. Spore crop preparation and enumeration. Stock spore cultures were maintained at -80 C as spore suspensions or in cooked-meat medium (Difco). Spore crops were prepared by the biphasic technique as described previously (19). Spores of C. botulinum strains 17409A, 25763, 62A, 56A, Clovis, 999, 169, 213, Aphis 4B, 53B, 2129, KAP (NFPA), 17B, and were used, as either a single pool or two pools containing either proteolytic or nonproteolytic strains. The spore crops were enumerated on BHI agar (Difco) poised with 0.05% filter-sterilized cysteine-hcl (Sigma). Unless otherwise indicated, spore suspensions in deionized distilled water were heat activated for 10 min at either 65 C (nonproteolytic strains) or 80 C (proteolytic strains). Bacteriocin production and detection. Bacteriocin production and detection were by the spot-on-the-lawn deferred (16) and simultaneous (19) antagonism methods. Overnight bacteriocinogenic and control cultures were harvested and washed twice by centrifugation at 4 C and 5,000 x g for 10 min. The cell pellets were resuspended in fresh MRS broth and standardized to an optical density (660 nm) of 0.40, which corresponded to a viable cell count of ca. 108 CFU/ml. Two-microliter volumes of the standardized cultures were spot inoculated onto basal BHI agar (0.5% NaCl) plates and BHI plates supplemented with NaCl to final concentrations of either 2.5 or 5%. The inoculated plates were incubated anaerobically in GasPak anaerobic jars (BBL Microbiology Systems, Cockeysville, Md.) for 48 h at 4, 10, 15, or 30 C. The plates were then transferred to the anaerobic chamber, overlaid as previously described (19) with soft basal BHI agar (0.7% Bacto Agar) seeded with approximately 1.4 x 104 CFU/ml of a single pool of proteolytic and nonproteolytic C. botulinum types A and B spores, and then incubated further for 24 h at 35 C. The simultaneous antagonism testing was as described above except that the standardized bacteriocinogenic cell suspensions were subjected to serial 100-fold dilutions in fresh MRS broth and spot inoculated onto indicator spore lawns prepared with 1.4 x 104, 1.4 x 105, or 1.4 x 106 CFU/ml of spore pool in basal BHI broth (which contains 0.5% NaCl) solidified with 1% agar and supplemented with NaCl to final concentrations of 3 or 4%. The spore pools consisted of either proteolytic type A and B or nonproteolytic type B strains. Each of the three indicator spore lawns, in Y-sectioned petri dishes, was challenged with 0 (MRS broth), 102, 104, and 106 CFU of bacteriocin-producing cells per ml of suspension. Plates seeded with the pool of proteolytic types A and B spores were incubated at 15 or 35 C, and the nonproteolytic type B plates were incubated at either 4 or 10 C in anaerobic jars. The plates were incubated for various times and checked for inhibition zones. Extraction of bacteriocins from agar cultures grown at refrigeration temperatures. Petri dishes containing 15 ml of BHI broth supplemented with 2.5% NaCl (final concentration) and solidified with 0.4% agar were spread inoculated with 400,ul of overnight cultures of L. lactis 11454, P. pentosaceus 43200, and L. plantarum BN. The plates were incubated anaerobically at either 4 or 10 C for 5 days and then aseptically transferred to sterile flasks and frozen at -20 C until needed. The agar cultures were thawed at room temperature for 3 to 4 h. After the freeze-thaw cycle broke the gel structure, chunks of agar were removed by being TABLE 1. Bacteriocinogenic strain Effects of initial growth temperature on inhibition of C. botulinum spores' Radius of inhibition zone (mm)b at the following incubation temp: 4 C 10 C 150C 300C L. lactis P. pentosaceus P. pentosaceus L. plantarum BN L. plantarum LB L. plantarum LB L. acidophilus N L. plantarum 8014C a Bacteriocin production and assay was by deferred antagonism method. The lawns (ca. 104 CFU/ml) were prepared with proteolytic and nonproteolytic strains. Bacteriocinogenic strains on BHI agar were grown for 48 h at the respective temperatures and then further incubated for 24 h at 35 C following overlay with seeded BHI agar. b Values are average radii with standard deviations of 0 to 0.3 (n = 4). +, inhibition zones of <0.5 mm. I Control, nonbacteriocinogenic strain. strained through double-layer sterile cheese cloth. Cells were removed by centrifugation of the filtrates at 10,000 x g for 10 min at 4 C and then by passage through 0.45-,um-poresize Acrodisc filters (Gelman Sciences, Ann Arbor, Mich.). The filtrates from L. plantarum BN, P. pentosaceus 43200, and L. lactis 11454, hereafter referred to as crude plantacin BN, pediocin A (4), and nisin (27) preparations, respectively, were assayed (100,ul per well) for activities at 35 C for 24 h by well diffusion methods (29) using either Lactobacillus sake or C. botulinum 169B spores (ca. 104 CFU/ml) to prepare the indicator lawns on Trypticase soy agar (without glucose) supplemented with 0.5% yeast extract. The ph of the crude agar-extracted bacteriocin preparations varied between 5.8 and 6.0 and was adjusted with NaOH to 6.5 prior to clarification and membrane sterilization. RESULTS APPL. ENVIRON. MICROBIOL. Deferred inhibition at refrigeration and abuse temperatures. The radii of growth inhibition zones were affected by the initial incubation temperatures (Table 1). The inhibition zones on lawns prepared with a composite inoculum of proteolytic and nonproteolytic spores increased with increasing incubation temperature. Bacteriocinogenic colonies at 4 and 30 C were, respectively, associated with the smallest and largest inhibition zones. This general trend was the same for all the strains included in the study. L. lactis was the most active and was followed by P. pentosaceus and and L. plantarum BN (Table 1). L. plantarum LB75 and LB592 did not produce bacteriocins at 4 and 10 C. L. acidophilus N2 was inactive at all the temperatures. L. plantarum 8014, the non-bacteriocin-producing strain, did not produce inhibition zones. The addition of sodium chloride to the production medium had a mixed effect on the bacteriocinogenicity of the strains. The radii of growth inhibition zones associated with L. lactis and L. plantarum BN increased with increasing salt concentrations (Fig. 1). The bacteriocinogenicity of all the other strains was not markedly affected by 5% NaCl in the production medium. Bacteriocin production and inhibition of nonproteolytic C. botulinum spores at 4 and 10 C by direct antagonism. L.

3 VOL. 57, 1991 BACTERIOCIN INHIBITION OF C. BOTULINUM SPORES z0 I a * BN -V sodium chloride concentration, % 5 6 A *@- LB592 LB75 N2 FIG. 1. Effects of sodium chloride in bacteriocin production medium on radii of inhibition zones. Each point is the average of four measurements, with a standard deviation of 0 to 0.1 mm. Bacteriocinogenicity was by deferred spot-on composite proteolytic and nonproteolytic C. botulinum types A and B lawn at 35 C. plantarum LB75 and LB592 and L. acidophilus N2 were not included in subsequent studies because of their apparent inactivity in the experiments described above. A pool of nonproteolytic type B spores [25765, 2129, 17B, and KAP (NFPA)] was used to prepare the lawns (ca. 104 CFU/ml) for the data of Table 2. The plates were incubated at either 4 C (Table 2) or 10 C for 60 days. Plates with poorly developed lawns were incubated further at 35 C to enhance lawn development. Increasing the bacteriocinogenic cell inoculum levels from 102 through 104 to 106 CFU/ml increased the radii of inhibition zones. L. plantarum BN and L. lactis TABLE 2. Effects of sodium chloride and cell concentration of bacteriocinogenic strains on inhibition of nonproteolytic type B spores at 4 C Bacteriocinogenic strain (log CFU/ml) Radius of inhibition zone (mm)' at the following NaCl concn: 0.5% 3.0% 4.0% L. lactis ND 1.1 NG 4 ND ND P. pentosaceus NG NG P. pentosaceus NG NG L. plantarum BN 2 ND 0.5 CI 4 ND 1.4 CI 6 ND 3.4 CI a Bacteriocin production and assay was by the simultaneous antagonism method. Values are average radii (standard deviations, 0 to 0.1; n = 4) of inhibition zones on lawns prepared with 104 CFU/ml of nonproteolytic spore pool in BHI agar. ND, not tested; NG, no growth of bacteriocin-producing cells; CI, indicator lawns completely inhibited even after further incubation at 35 C. TABLE 3. Effects of sodium chloride and cell concentration of bacteriocinogenic strains on inhibition of proteolytic types A and B spores at 15 C Bacteriocinogenic strain (log CFU/ml) Radius of growth inhibition zone (mm)a at the following NaCI concn: 0.5% 3.0% 4.0% L. lactis CI CI CI 4 CI CI CI 6 CI CI CI P. pentosaceus CI CI CI P. pentosaceus CI CI CI L. plantarum BN 2 ND CI CI 4 ND CI CI 6 ND CI CI a Bacteriocin production and assay was by the simultaneous antagonism method. Values are average radii (standard deviations, 0 to 0.3; n = 4) of growth inhibition zones on lawns prepared with 104 CFU/ml of pooled proteolytic strains of C. botulinum spores in BHI agar. CI, indicator lawns completely inhibited even after further incubation at 35 C; ND, not determined. were more active than P. pentosaceus and Inhibition zone sizes decreased when increasing levels of C. botulinum spores were used in the lawn (data not shown). Again, sodium chloride had a mixed effect on the growth and bacteriocinogenicity of the strains. Its influence depended on the inoculum levels of the bacteriocinogenic cultures. Bacteriocinogenicity increased with increasing sodium chloride concentration when a 106-CFU/ml inoculum of the bacteriocinogenic strain was used. Three and 4% sodium chloride inhibited the growth of 102 CFU of P. pentosaceus and and L. lactis per ml. Salt stimulated L. plantarum BN growth and bacteriocin production at all inoculum levels. The botulinal spores in the indicator lawns on 4% NaCl-supplemented BHI production agar were completely inhibited by L. plantarum BN even after further incubation at 35 C for 7 days. Sodium chloride was less inhibitory to the growth and bacteriocinogenicity of the bacteriocinogenic cells at 10 C. All the strains, at all three inoculum levels and sodium chloride concentrations in the production medium, completely inhibited indicator lawns prepared with 104 CFU of botulinal spores per ml (data not shown). Inhibition of proteolytic C. botulinum spores at 15 and 35 C by simultaneous antagonism. The data in Table 3 were obtained from lawns prepared with 104 CFU of spores of C. botulinum types A and proteolytic B strains (17409A, 25763, 62A, 56A, Clovis, 999, 169, 213, Aphis 4B, and 53B) per ml and incubated at 15 C for 45 days. All the bacteriocinogenic strains grew at 15 C and were not inhibited by NaCl. Growth inhibition zone sizes increased with increasing salt and bacteriocinogenic inoculum levels. The C. botulinum lawns were completely inhibited by all bacteriocinogenic cell concentrations in the presence of 4% sodium chloride. At 35 C inhibition zone sizes increased with increasing

4 3426 OKEREKE AND MONTVILLE APPL. ENVIRON. MICROBIOL. I I 6 8 o 2 A log cell (CFUI ml) FIG. 2. Sodium chloride potentiation of antibotulinal activities of L. plantarum BN (A), P. pentosaceus (B), L. lactis (C), and P. pentosaceus (D). Each point is the average of four measurements, with a standard deviation of 0 to 0.4 mm. Bacteriocinogenicity was by simultaneous spot-on composite C. botulinum types A and proteolytic B lawn at 35 C. bacteriocinogenic cell concentrations at all salt levels in the production medium (Fig. 2). The plates were incubated for 3 days. Plates containing 4% NaCl in the production medium were incubated further for 1 to 11 days. Inhibition of botulinal spores by all the bacteriocinogenic strains was increased by 4% sodium chloride (Fig. 2). Although 102- and 104-CFU/ml inocula of P. pentosaceus were sufficient for colony formation, no inhibition zones were associated with these colonies in the presence of 0.5 and 3% NaCl. Zone sizes decreased with increasing spore concentrations in the indicator lawns (data not shown). Extraction of bacteriocin from cultures grown at 4 and 10 C. Neither plantacin BN nor nisin extracted from the 4 C preparations produced inhibition zones on lawns prepared with C. botulinum spores (data not shown). But the 10 C crude nisin preparations produced an average radius of inhibition zone of 1.2 mm. When L. sake 15521, which is more sensitive than C. botulinum to bacteriocins, was used in the bacteriocin assays, we could detect bacteriocin activity from extracts of L. plantarum BN and L. lactis agar cultures that had been grown at refrigeration temperatures (Table 4). Nisin and plantacin BN production were greater at 10 C than at 4 C, as demonstrated by well diffusion assays of crude bacteriocin preparations from freeze-thawed agar cultures (Table 4). Effects of sodium chloride on C. botulinum spores. The effects of sodium chloride on the viability of the spore inoculum used to prepare the indicator lawns were determined on basal BHI agar supplemented with 1.5, 3.5, and 4.5% NaCl (final concentrations). The results in Table 5 suggest that viability of the spores was slightly affected by 4.5% NaCl in the enumeration medium. However, decreasing the spore level twofold did not result in marked increases in zone size (data not shown). TABLE 4. Bacteriocin preparation' Bacteriocin production at refrigeration temperatures by bacteriocinogenic strains Mean radius of inhibition zone (mm)b at the following incubation temp: 4 C 1O C Plantacin BN 2.76 ± ± 0.36 Nisin 5.64 ± ± 0.62 Pediocin A 0 0 a Samples were obtained by freeze-thawing of agar cultures of the bacteriocinogenic strains and assayed for bacteriocin activity by well diffusion. Indicator lawns were prepared with 104 CFU of L. sake per ml. b Values represent mean ± standard deviation; n = 10.

5 VOL. 57, 1991 BACTERIOCIN INHIBITION OF C. BOTULINUM SPORES 3427 TABLE 5. Effects of sodium chloride on colony formation by C. botulinum spores' % NaCI added to basal BHI CFU/ml (107) % Recovery agarb l a The pool of proteolytic strains was used. The plates were incubated at 35 C for 3 days. b Basal BHI agar contains 0.5% NaCl. Basal agar CFU per milliliter was regarded as 100% recovery. DISCUSSION The results presented here demonstrate the bacteriocinogenicity of L. lactis 11454, L. plantarum BN, and P. pentosaceus and at refrigeration and abuse temperatures against proteolytic and nonproteolytic C. botulinum spores. Bacteriocins were produced at refrigeration temperatures, but zone sizes increased with increasing incubation temperatures. Apparently the bacteriocinogenic cells were under less stress and grew optimally (more dense and larger colonies) at the higher temperatures, which consequently led to the production of more bacteriocins, as indicated by larger zone sizes (Table 1). That nisin and plantacin BN were produced during the period of refrigerated incubation and not coincident with the 35 C lawn development was confirmed in experiments in which the bacteriocinogenic strains were inoculated on dialysis membranes placed on the agar. When these were removed after the 4, 10, or 15 C incubation prior to the transfer to 35 C, inhibition zones were still produced (data not shown). The extraction of nisin and plantacin BN from cultures grown at refrigeration temperatures (Table 4) shows unequivocally that the bacteriocins were produced at the refrigeration temperatures prior to transfer of the plates to the 35 C anaerobic chamber. Because L. sake is more bacteriocin sensitive than C. botulinum spores (22), it was used to assay crude plantacin BN, pediocin A, and nisin. Although plantacin BN and nisin produced inhibition zones of 3 and 9 mm, respectively, on L. sake lawns, the same bacteriocin preparations were barely inhibitory to C. botulinum spores (data not shown). Although the lower growth limit for proteolytic C. botulinum is 10 to 12 C (14) and 3.3 to 4 C for nonproteolytic strains of types B and F (5, 14), lawns prepared with ca. 104 CFU of nonproteolytic type B spores per ml were completely inhibited by low inocula of all the bacteriocinogenic strains at 10 C. At 15 and 35 C indicator lawns of proteolytic types A and B spores developed faster, especially on the basal production medium. However, the bacteriocinogenic cells were less inhibited by the high salt concentrations, resulting in increased zones of inhibition. The 15 and 35 C studies were included to simulate temperature abuse conditions of minimally processed refrigerated meat products. The results suggest that at 15 C and 4% NaCl the bacteriocinogenic strains included in the study will produce sufficient bacteriocins to inhibit spore outgrowth. At 35 C, the optimum C. botulinum growth temperature, outgrowth inhibition was limited even in the presence of 4% NaCl. The results indicated that supplementation of BHI with sodium chloride (3 and 4%) either potentiated antibotulinal bacteriocin activities at 10, 15, and 35 C or made the indicator spores more bacteriocin sensitive. The data in Table 5 were an attempt to address the second possibility. The results indicate that the viabilities of botulinal spores were in the same order of magnitude at all salt concentrations and that supplementation of basal BHI agar with 4.5% NaCl resulted in less than 1 log cycle reduction in the number of spores that formed colonies. The radii of inhibition zones are exponentially related to the inoculum levels of indicator cells (15). Hence, doubling of cell concentration in indicator lawns does not result in halving of the radii of inhibition zones by a given concentration of nisin (unpublished data). It appeared, therefore, that NaCl diffused from the bacteriocin production medium to the detection agar and potentiated bacteriocin activity against the spores. The fact that the inhibitory effects of sodium chloride were evident only under deferred (Fig. 1) and not under simultaneous (Tables 2 and 3; Fig. 2) antagonism supports this conclusion. Although spores of proteolytic strains of C. botulinum have been reported to exhibit high resistance to salt and a, (10), our results suggest that the combined effect of the bacteriocins and NaCl was a reduction in the resistance of the spores and increased inhibition of outgrowth. This agrees with the observation that other inhibitory compounds act synergistically with nisin (13). Contrary to literature reports (1, 23) that high cell numbers of bacteriocinogenic strains are required for bacteriocinmediated inhibition of pathogens, our results indicate a complete inhibition of indicator lawns by 102 CFU of bacteriocin-producing cells per ml. We have demonstrated in this study bacteriocin-mediated inhibition of relatively large numbers of C. botulinum spores by bacteriocinogenic strains of the lactic acid bacteria. The results also demonstrated bacteriocin production at refrigeration temperatures and enhancement of antibotulinal activities by 3 and 4% NaCl in the production media. To our knowledge, this is the first report of bacteriocin production at refrigeration temperatures. 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