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Antimicrobial activity of enterocin EJ97 on Bacillus coagulans CECT 12
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 20 (2003) 533–536
www.elsevier.nl/locate/jnlabr/yfmic
Antimicrobial activity of enterocin EJ97 on Bacillus coagulans CECT 12 M.T. Garc!ıa, N. Ben Omar, R. Lucas, R. Pe! rez-Pulido, A. Castro, M.J. Grande, * M. Mart!ınez-Canamero, A. Ga! lvez* ! Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Area de Microbiolog!ıa, Universidad de Ja!en., Jaen 23071, Spain Received 18 September 2002; accepted 15 November 2002
Abstract Enterocin EJ97 showed a concentration-dependent bactericidal activity against vegetative cells of Bacillus coagulans CECT 12. Highest activity was obtained at incubation temperatures of 371C and 151C, but almost no activity was detected during incubation of cells at 41C. Enterocin EJ97 showed highest activity against B. coagulans CECT 12 at pH 7.0. Some decrease of activity was detected at pH 5.0, and a very low activity was detected during incubation of cells at pH 9.0. Enterocin activity was not modified by sodium benzoate or by sodium chloride, but it was enhanced to some extent by sodium tripolyphosphate, and was highly potentiated by sodium nitrite at concentrations above 50 mg/ml. r 2003 Elsevier Ltd. All rights reserved. Keywords: Bacteriocin; Enterocin; Bacillus coagulans
1. Introduction The contamination of food products with endosporeforming bacteria is a common problem in the food industry, because endospores require more intense treatments for inactivation than vegetative cells. This results in higher processing costs and less preserved product quality. Risks associated to endospore survival during processing are specially significant in canned foods, and acidification is often used as an additional hurdle to prevent microbial growth and spoilage. Bacillus coagulans is a mild acidophilic endosporeforming bacterium of significance in vegetable foods (Brackett, 2001) and in canned fruits, especially in tomato products. This bacterium is also frequently found in milk and some dairy products (Cosentino et al., 1997, Shehata et al., 1983), and has been involved in alteration of evaporated milk (Kalogridou-Vassiliadou, 1992) and canned condensed milk (Caric! , 1994), causing economic losses because of unwanted acidification and/ or coagulation. B. coagulans has also been described *Corresponding author. Tel.: +34-953-0212-160; fax: +34-9530212-141. E-mail address: [email protected] (A. G!alvez). 0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00167-3
among the bacteria involved in cork taint spoilage of wine (Sponholz, 1993). In recent years, the use of naturally occurring antimicrobial substances in the control of spoilage micro-organisms has gained great attention (Stiles, 1996), motivated by the demands of consumers for foods that are safer, naturally preserved and processed by milder treatments. Bacteriocins are antimicrobial substances of ribosomal synthesis produced by many different types of bacteria. They show diverse spectra of activity, but the producer strains are always resistant (Jack et al., 1995). Enterocin EJ97 is a low-molecular weight bacteriocin produced by Enterococcus faecalis EJ97 (Ga! lvez et al., 1998). The complete amino acid sequence and the genetic determinants of EJ97 have been recently elucidated (Sa! nchez-Hidalgo et al., 2002) and indicate that this is a novel bacteriocin synthesized without leader peptide. Enterocin EJ97 shows antimicrobial activity against bacteria involved in food spoilage (B. coagulans, B. stearothermophilus) and food-poisoning (Listeria monocytogenes, Staphylococcus aureus) (Ga! lvez et al., 1998). The effect of enterocin EJ97 on B. coagulans and the influence of external factors on bacteriocin activity are described in this work.
ARTICLE IN PRESS 534
M.T. Garc!ıa et al. / Food Microbiology 20 (2003) 533–536
2. Methods 2.1. Bacterial strains and culture conditions
Table 1 Effect of enterocin EJ97 alone or in combination with chemical preservatives on cell viability of B. coagulans CECT 12 Chemical preservative
B. coagulans CECT 12 was obtained from the Spanish ! Espanola * Type Culture Collection (Coleccion de Cultivos Tipo, CECT). E. faecalis S-47 was used as an indicator strain for determination of bacteriocin activity. Bacteria were propagated in brain–heart infusion broth (BHI; Scharlau, Barcelona) at 371C. 2.2. Source of enterocin EJ97 The bacteriocin EJ97 was obtained from cultured broths of the producer strain by cation exchange chromatography as described elsewhere (Ga! lvez et al., 1998). Samples showing bacteriocin activity were dialysed overnight through a low-Mw cut-off dialysis tubing (Sigma), and then filtrated through 0.22 mm filters (Millipore). The protein concentration of samples was determined by the method of Bradford (Bradford, 1976). Bacteriocin concentrates were diluted serially in sterile saline solution and tested against the indicator strain E. faecalis S-47 for antimicrobial activity, using the agar-well diffusion assay (Tagg and McGiven, 1971). One arbitrary unit (AU) was defined as the highest dilution that produced a visible halo of inhibition. The bacteriocin concentration of samples was determined from the specific activity value of 1.60 AU/mg of protein obtained for pure bacteriocin (Ga! lvez et al., 1998). 2.3. Determination of antimicrobial activity on vegetative cells Exponential-phase cultures of B. coagulans CECT 12 growing in BHI broth at 371C were diluted in prewarmed BHI broth containing different concentrations of enterocin EJ97. At different intervals of incubation at the desired temperature, cultures were serially diluted in sterile saline solution and plated in triplicate on tryptic soy agar (TSA), and the plates were incubated at 371C during 48 h for viable cell counts. Incubation at 151C was done in a refrigerated incubation chamber (Memmert). Incubation at 41C was done in a conventional refrigeration chamber. Cultures were kept at the desired temperature for 30 min before addition of bacteriocin. In order to test the effect of pH on antimicrobial activity, enterocin EJ97 was added to exponential-phase cultures inoculated in BHI broth adjusted to pH 5.0 with diluted HCl acid, or adjusted to pH 9.0 with NaOH. The chemical preservatives listed in Table 1 were added to BHI broth at the desired concentration, and the pH was readjusted to neutrality if necessary.
None Na-Benzoate (0.05%) Na-Benzoate (0.1%) NaCl (3%) NaCl (6%) Na-Tripolyphosphate (0.3%) Na-Tripolyphosphate (0.5%) Na-Nitrite (25 mg/ml) Na-Nitrite (50 mg/ml) Na-Nitrite (100 mg/ml)
Enterocin EJ97a 0 mg/ml
1 mg/ml
6.2070.43 6.0070.81 5.2370.72 6.6070.67 6.4670.49 6.2770.54 5.6570.39 5.9570.62 4.5470.39 3.6070.54
4.2770.31 4.9570.50 4.8470.32 4.2170.54 4.2370.71 4.1370.45 3.2370.54 4.1870.39 3.0070.25 070.10
Cells (ca. 5.60 log cfu/ml) were incubated in BHI broth with different chemical preservatives either alone or in combination with enterocin EJ97. The log number of viable cell concentrations after 4 h of incubation at 371C are shown. a Data represent the means of three independent determinations, 7 standard deviation (SD).
All experiments were performed in triplicate. The mean values of the data are presented.
3. Results and discussion 3.1. Effect of enterocin EJ97 on B. coagulans CECT 12, and influence of culture conditions on bacteriocin activity Enterocin EJ97 showed a concentration-dependent bactericidal activity on vegetative cells of B. coagulans CECT 12 incubated at 371C (Fig. 1A). At a final concentration of 1 mg/ml, the number of viable cells decreased during the first 4 h of incubation, and culture growth was partially inhibited after 24 h. Viable cell counts decreased noticeably for a bacteriocin concentration of 2 mg/ml, and no viable cells were detected after 4 h of incubation with 3 mg/ml. The bactericidal activity of EJ97 against B. coagulans CECT 12 was not modified when the cultures were incubated at 151C (Fig. 1B). At this temperature, growth of controls was slower compared to 371C, but the reductions in the numbers of viable cells obtained after bacteriocin treatment were almost the same, in the sense that similar bacteriocin concentrations showed similar bactericidal effects. Enterocin activity on cultures incubated at 41C was markedly reduced, since no bactericidal effects were observed with bacteriocin concentrations of up to 4 mg/ ml (Fig. 1C). At 8 mg/ml, a reduction of ca. two log units was observed during the first 12 h of incubation, but the viability of the remaining cells was not affected during further incubation for up to 48 h. Altogether, these results suggest that enterocin EJ97 only kills actively
ARTICLE IN PRESS M.T. Garc!ıa et al. / Food Microbiology 20 (2003) 533–536
535
Fig. 1. Effect of enterocin EJ97 on exponential-phase cultures of B. coagulans CET 12 in BHI broth incubated at 371C (A), 151C (B), and 41C (C). Cultures were treated with final bacteriocin concentrations of 0 (J), 1 (m), 2 () and 3 mg/ml (’), (panels A and B) or with 0 (J), 2 (m), 4 () and 8 mg/ml (’), (panel C).
growing cells. Therefore, growth of B. coagulans in foods kept at ambient temperature (like, for example, canned foods) could be inhibited by enterocin EJ97. Nevertheless, the efficacy of this bacteriocin and its interaction with food components should be tested in situ. The influence of pH on bacteriocin activity was tested in BHI broth adjusted to pH values of 5.0, 7.0 and 9.0, using a bacteriocin concentration of 2 mg/ml. Highest activity was detected at pH 7.0 (Fig. 2). Comparatively, activity was lower at pH 5.0, as shown by the slower decline in the number of viable cells observed during incubation. The effect of EJ97 was much more limited at pH 9.0. For the same bacteriocin concentration, no effect was observed on the initial numbers of viable cells, although the growth of cultures in the presence of EJ97 was much slower than in the untreated controls. These results suggest that enterocin EJ97 is less active at alkaline pH or that the test strain is less sensitive under these conditions. Previous studies showed that enterocin EJ97 was completely stable in the pH range of 2.0–9.5 (Ga! lvez et al., 1998). Because foods more frequently spoiled by B. coagulans are low-acid canned foods (like fruits and vegetables) and neutral pH foods (like evaporated or condensed milk), the pH-dependent profile of sensitivity of B. coagulans to enterocin EJ97 should be compatible with such food environments as far as pH concerns.
Fig. 2. Influence of pH on the activity of enterocin EJ97 against B. coagulans CET 12. Cultures were incubated at 371C in BHI broth adjusted to pH 5.0 (m), 7.0 () or 9.0 (’) without bacteriocin (open symbols) or with EJ97 at a final concentration of 2 mg/ml (closed symbols).
3.2. Influence of chemical preservatives on the activity of enterocin EJ97 against B. coagulans CECT 12 Chemical preservatives used in foods as additional hurdles for preservation may act as potentiators or inhibitors of bacteriocin activity (G.anzle et al., 1998). Therefore, the activity of enterocin EJ97 was tested at sub-inhibitory concentrations (1 mg/ml) against vegetative cells of B. coagulans CECT 12 (4 105 cfu/ml) in the presence of four different chemical preservatives (Table 1). The sodium salt of benzoic acid had a slight
ARTICLE IN PRESS 536
M.T. Garc!ıa et al. / Food Microbiology 20 (2003) 533–536
inhibitory effect on bacterial growth, that was proportional to the final concentration tested. At 0.1%, sodium benzoate caused a noticeable delay on growth, but it showed no effect on cell viability. The combination of EJ97 and sodium benzoate at concentrations of 0.05% and 0.1% had a similar effect on bacterial growth as the addition of bacteriocin alone, indicating that this compound did not have any potentiating or antagonizing effect on enterocin activity. Sodium chloride had no effect on growth of B. coagulans CECT 12 at the concentrations tested (3% and 6%). Furthermore, bacteriocin activity was not modified at all by the presence of sodium chloride. The chelating agent sodium tripolyphosphate had no effect on bacterial growth at a final concentration of 0.3%. At 0.5%, some delay of growth was detected (as shown by the lower number of viable cells at 4 h of incubation in comparison with the untreated control). Sodium tripolyphosphate had no effect on the activity of enterocin EJ97 at a final concentration of 0.3%. However, when the chelating agent was tested at 0.5% in combination with EJ97, the number of viable cells was ca. one log unit lower compared to cultures treated with EJ97 alone, indicating a potentiating effect. Sodium nitrite showed a clear inhibitory effect on bacterial growth as well as cell viability, depending on the final concentration tested. After 4 h of incubation, a marked reduction in the initial concentrations of viable cells was observed for nitrite concentrations of 50 mg/ml or above. Upon incubation with sodium nitrite (50 mg/ ml) and enterocin EJ97 (1 mg/ml) viable cell counts were one log unit lower compared to cultures incubated with bacteriocin alone. The combination of enterocin EJ97 and sodium nitrite (100 mg/ml) killed all viable cells of B. coagulans CECT 12 within 4 h of incubation at 371C, indicating a synergistic action of the bacteriocin with nitrite. Activity of enterocin against L. monocytogenes is also enhanced by nitrite (unpublished results). The antimicrobial activity of other bacteriocins like enterocin AS-48 (Abriouel et al., 2002), nisin (Shahidi, 1991) or lactocin 705 (Vignolo et al., 1998) can also be enhanced by this chemical compound. Although sodium nitrite is only permitted as additive for meat products (with residual limits ranging from 50 to 175 mg/kg; European Parliament and Council Directive No. 95/2/ EC and amendments), nitrites are also found naturally in vegetable foods, and canned vegetable juices have been reported to exceed the acceptable daily intake of nitrite (Cheng and Tsang, 1998). Therefore, the influence of nitrite concentration on the activity of enterocin EJ97 in nitrite-containing foods should be studied. There are scarce data on the effects of bacteriocins against B. coagulans, although the antibiotic nisin is highly active both on vegetative cells and spores of this bacterium (Roberts and Hoover, 1996). There is a
growing interest on the combined use of bacteriocins in order to avoid selection for resistant strains. Therefore, the combined effects of nisin and enterocin EJ97 should be studied in vitro and in model food systems.
References Abriouel, H., Maqueda, M., G!alvez, A., Mart!ınez-Bueno, M., Valdivia, E., 2002. Inhibition of bacterial growth, enterotoxin production, and spore outgrowth in strains of Bacillus cereus by bacteriocin AS-48. Appl. Environ. Microbiol. 68, 1473–1477. Brackett, R.E., 2001. Fruits, vegetables, and grains. In: Doyle, M., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology, Fundamentals and Frontiers. ASM Press, Washington, DC, pp. 127–138. Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Cari!c, M., 1994. Concentrated and Dried Dairy Products. VCH Publ. Inc., New York. Cheng, C.F., Tsang, C.W., 1998. Simultaneous determination of nitrite, nitrate and ascorbic acid in canned vegetable juices by reverse-phase ion-interaction HPLC. Food Additives Contam. 15, 753–758. Cosentino, S., Mulargia, A.F., Pisano, B., Tuveri, P., Palmas, F., 1997. Incidence and biochemical characteristics of Bacillus flora in Sardinian dairy products. Int. J. Food Microbiol. 38, 235–238. G!alvez, A., Valdivia, E., Abriouel, H., Camafeita, E., M!endez, E., Mart!ınez-Bueno, M., Maqueda, M., 1998. Isolation and characterization of enterocin EJ 97, a bacteriocin produced by Enterococcus faecalis EJ 97. Arch. Microbiol. 171, 59–65. G.anzle, M.G., Weber, S., Hammes, W.P., 1998. Effect of ecological factors on the inhibitory spectrum and activity of bacteriocins. Int. J. Food Microbiol. 46, 207–217. Jack, R.W., Tagg, J.R., Ray, B., 1995. Bacteriocins of gram-positive bacteria. Microbiol. Rev. 59, 171–200. Kalogridou-Vassiliadou, D., 1992. Biochemical activities of Bacillus species isolated from flat sour evaporated milk. J. Dairy Sci. 75, 2681–2686. Roberts, C.M., Hoover, D.G., 1996. Sensitivity of Bacillus coagulans spores to combinations of high hydrostatic pressure, heat, acidity and nisin. J. Appl. Bacteriol. 81, 363–368. S!anchez-Hidalgo, M., Maqueda, M., G!alvez, A., Abriouel, H., Valdivia, E., Mart!ınez-Bueno, M., 2002. The protein sequence and genetic determinants of enterocin EJ. Book of Abstracts. International Symposium on Enterococci in Foods—Functional and Safety Aspects, Berlin. Shahidi, F., 1991. Developing alternative meat-curing systems. Trends Food Sci. Technol. 2, 219–222. Shehata, A.E., Magdoub, M.N., Sultan, N.E., El-Samgray, Y.A., 1983. Aerobic mesophilic and psychrotrophic sporeforming bacteria in buffalo milk. J. Dairy Sci. 66, 1228–1231. Sponholz, W.R., 1993. Wine spoilage by microorganisms. In: Fleet, G.H. (Ed.), Wine Microbiology, Biotechnology. Harwood Academy Publications, Chur, Switzerland, pp. 395–420. Stiles, M.E., 1996. Biopreservation by lactic acid bacteria. Antonie van Leeuwenhoek 70, 221–245. Tagg, J.R., McGiven, R., 1971. Assay system for bacteriocins. Appl. Microbiol. 21, 943. Vignolo, G., Fadda, S., de Kairuz, M.N., de R Holgado, A.P., Oliver, G., 1998. Effects of curing additives on the control of Listeria monocytogenes by lactocin in meat slurry. Food Microbiol. 15, 259–264.
www.elsevier.nl/locate/jnlabr/yfmic
Antimicrobial activity of enterocin EJ97 on Bacillus coagulans CECT 12 M.T. Garc!ıa, N. Ben Omar, R. Lucas, R. Pe! rez-Pulido, A. Castro, M.J. Grande, * M. Mart!ınez-Canamero, A. Ga! lvez* ! Departamento de Ciencias de la Salud, Facultad de Ciencias Experimentales, Area de Microbiolog!ıa, Universidad de Ja!en., Jaen 23071, Spain Received 18 September 2002; accepted 15 November 2002
Abstract Enterocin EJ97 showed a concentration-dependent bactericidal activity against vegetative cells of Bacillus coagulans CECT 12. Highest activity was obtained at incubation temperatures of 371C and 151C, but almost no activity was detected during incubation of cells at 41C. Enterocin EJ97 showed highest activity against B. coagulans CECT 12 at pH 7.0. Some decrease of activity was detected at pH 5.0, and a very low activity was detected during incubation of cells at pH 9.0. Enterocin activity was not modified by sodium benzoate or by sodium chloride, but it was enhanced to some extent by sodium tripolyphosphate, and was highly potentiated by sodium nitrite at concentrations above 50 mg/ml. r 2003 Elsevier Ltd. All rights reserved. Keywords: Bacteriocin; Enterocin; Bacillus coagulans
1. Introduction The contamination of food products with endosporeforming bacteria is a common problem in the food industry, because endospores require more intense treatments for inactivation than vegetative cells. This results in higher processing costs and less preserved product quality. Risks associated to endospore survival during processing are specially significant in canned foods, and acidification is often used as an additional hurdle to prevent microbial growth and spoilage. Bacillus coagulans is a mild acidophilic endosporeforming bacterium of significance in vegetable foods (Brackett, 2001) and in canned fruits, especially in tomato products. This bacterium is also frequently found in milk and some dairy products (Cosentino et al., 1997, Shehata et al., 1983), and has been involved in alteration of evaporated milk (Kalogridou-Vassiliadou, 1992) and canned condensed milk (Caric! , 1994), causing economic losses because of unwanted acidification and/ or coagulation. B. coagulans has also been described *Corresponding author. Tel.: +34-953-0212-160; fax: +34-9530212-141. E-mail address: [email protected] (A. G!alvez). 0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0740-0020(02)00167-3
among the bacteria involved in cork taint spoilage of wine (Sponholz, 1993). In recent years, the use of naturally occurring antimicrobial substances in the control of spoilage micro-organisms has gained great attention (Stiles, 1996), motivated by the demands of consumers for foods that are safer, naturally preserved and processed by milder treatments. Bacteriocins are antimicrobial substances of ribosomal synthesis produced by many different types of bacteria. They show diverse spectra of activity, but the producer strains are always resistant (Jack et al., 1995). Enterocin EJ97 is a low-molecular weight bacteriocin produced by Enterococcus faecalis EJ97 (Ga! lvez et al., 1998). The complete amino acid sequence and the genetic determinants of EJ97 have been recently elucidated (Sa! nchez-Hidalgo et al., 2002) and indicate that this is a novel bacteriocin synthesized without leader peptide. Enterocin EJ97 shows antimicrobial activity against bacteria involved in food spoilage (B. coagulans, B. stearothermophilus) and food-poisoning (Listeria monocytogenes, Staphylococcus aureus) (Ga! lvez et al., 1998). The effect of enterocin EJ97 on B. coagulans and the influence of external factors on bacteriocin activity are described in this work.
ARTICLE IN PRESS 534
M.T. Garc!ıa et al. / Food Microbiology 20 (2003) 533–536
2. Methods 2.1. Bacterial strains and culture conditions
Table 1 Effect of enterocin EJ97 alone or in combination with chemical preservatives on cell viability of B. coagulans CECT 12 Chemical preservative
B. coagulans CECT 12 was obtained from the Spanish ! Espanola * Type Culture Collection (Coleccion de Cultivos Tipo, CECT). E. faecalis S-47 was used as an indicator strain for determination of bacteriocin activity. Bacteria were propagated in brain–heart infusion broth (BHI; Scharlau, Barcelona) at 371C. 2.2. Source of enterocin EJ97 The bacteriocin EJ97 was obtained from cultured broths of the producer strain by cation exchange chromatography as described elsewhere (Ga! lvez et al., 1998). Samples showing bacteriocin activity were dialysed overnight through a low-Mw cut-off dialysis tubing (Sigma), and then filtrated through 0.22 mm filters (Millipore). The protein concentration of samples was determined by the method of Bradford (Bradford, 1976). Bacteriocin concentrates were diluted serially in sterile saline solution and tested against the indicator strain E. faecalis S-47 for antimicrobial activity, using the agar-well diffusion assay (Tagg and McGiven, 1971). One arbitrary unit (AU) was defined as the highest dilution that produced a visible halo of inhibition. The bacteriocin concentration of samples was determined from the specific activity value of 1.60 AU/mg of protein obtained for pure bacteriocin (Ga! lvez et al., 1998). 2.3. Determination of antimicrobial activity on vegetative cells Exponential-phase cultures of B. coagulans CECT 12 growing in BHI broth at 371C were diluted in prewarmed BHI broth containing different concentrations of enterocin EJ97. At different intervals of incubation at the desired temperature, cultures were serially diluted in sterile saline solution and plated in triplicate on tryptic soy agar (TSA), and the plates were incubated at 371C during 48 h for viable cell counts. Incubation at 151C was done in a refrigerated incubation chamber (Memmert). Incubation at 41C was done in a conventional refrigeration chamber. Cultures were kept at the desired temperature for 30 min before addition of bacteriocin. In order to test the effect of pH on antimicrobial activity, enterocin EJ97 was added to exponential-phase cultures inoculated in BHI broth adjusted to pH 5.0 with diluted HCl acid, or adjusted to pH 9.0 with NaOH. The chemical preservatives listed in Table 1 were added to BHI broth at the desired concentration, and the pH was readjusted to neutrality if necessary.
None Na-Benzoate (0.05%) Na-Benzoate (0.1%) NaCl (3%) NaCl (6%) Na-Tripolyphosphate (0.3%) Na-Tripolyphosphate (0.5%) Na-Nitrite (25 mg/ml) Na-Nitrite (50 mg/ml) Na-Nitrite (100 mg/ml)
Enterocin EJ97a 0 mg/ml
1 mg/ml
6.2070.43 6.0070.81 5.2370.72 6.6070.67 6.4670.49 6.2770.54 5.6570.39 5.9570.62 4.5470.39 3.6070.54
4.2770.31 4.9570.50 4.8470.32 4.2170.54 4.2370.71 4.1370.45 3.2370.54 4.1870.39 3.0070.25 070.10
Cells (ca. 5.60 log cfu/ml) were incubated in BHI broth with different chemical preservatives either alone or in combination with enterocin EJ97. The log number of viable cell concentrations after 4 h of incubation at 371C are shown. a Data represent the means of three independent determinations, 7 standard deviation (SD).
All experiments were performed in triplicate. The mean values of the data are presented.
3. Results and discussion 3.1. Effect of enterocin EJ97 on B. coagulans CECT 12, and influence of culture conditions on bacteriocin activity Enterocin EJ97 showed a concentration-dependent bactericidal activity on vegetative cells of B. coagulans CECT 12 incubated at 371C (Fig. 1A). At a final concentration of 1 mg/ml, the number of viable cells decreased during the first 4 h of incubation, and culture growth was partially inhibited after 24 h. Viable cell counts decreased noticeably for a bacteriocin concentration of 2 mg/ml, and no viable cells were detected after 4 h of incubation with 3 mg/ml. The bactericidal activity of EJ97 against B. coagulans CECT 12 was not modified when the cultures were incubated at 151C (Fig. 1B). At this temperature, growth of controls was slower compared to 371C, but the reductions in the numbers of viable cells obtained after bacteriocin treatment were almost the same, in the sense that similar bacteriocin concentrations showed similar bactericidal effects. Enterocin activity on cultures incubated at 41C was markedly reduced, since no bactericidal effects were observed with bacteriocin concentrations of up to 4 mg/ ml (Fig. 1C). At 8 mg/ml, a reduction of ca. two log units was observed during the first 12 h of incubation, but the viability of the remaining cells was not affected during further incubation for up to 48 h. Altogether, these results suggest that enterocin EJ97 only kills actively
ARTICLE IN PRESS M.T. Garc!ıa et al. / Food Microbiology 20 (2003) 533–536
535
Fig. 1. Effect of enterocin EJ97 on exponential-phase cultures of B. coagulans CET 12 in BHI broth incubated at 371C (A), 151C (B), and 41C (C). Cultures were treated with final bacteriocin concentrations of 0 (J), 1 (m), 2 () and 3 mg/ml (’), (panels A and B) or with 0 (J), 2 (m), 4 () and 8 mg/ml (’), (panel C).
growing cells. Therefore, growth of B. coagulans in foods kept at ambient temperature (like, for example, canned foods) could be inhibited by enterocin EJ97. Nevertheless, the efficacy of this bacteriocin and its interaction with food components should be tested in situ. The influence of pH on bacteriocin activity was tested in BHI broth adjusted to pH values of 5.0, 7.0 and 9.0, using a bacteriocin concentration of 2 mg/ml. Highest activity was detected at pH 7.0 (Fig. 2). Comparatively, activity was lower at pH 5.0, as shown by the slower decline in the number of viable cells observed during incubation. The effect of EJ97 was much more limited at pH 9.0. For the same bacteriocin concentration, no effect was observed on the initial numbers of viable cells, although the growth of cultures in the presence of EJ97 was much slower than in the untreated controls. These results suggest that enterocin EJ97 is less active at alkaline pH or that the test strain is less sensitive under these conditions. Previous studies showed that enterocin EJ97 was completely stable in the pH range of 2.0–9.5 (Ga! lvez et al., 1998). Because foods more frequently spoiled by B. coagulans are low-acid canned foods (like fruits and vegetables) and neutral pH foods (like evaporated or condensed milk), the pH-dependent profile of sensitivity of B. coagulans to enterocin EJ97 should be compatible with such food environments as far as pH concerns.
Fig. 2. Influence of pH on the activity of enterocin EJ97 against B. coagulans CET 12. Cultures were incubated at 371C in BHI broth adjusted to pH 5.0 (m), 7.0 () or 9.0 (’) without bacteriocin (open symbols) or with EJ97 at a final concentration of 2 mg/ml (closed symbols).
3.2. Influence of chemical preservatives on the activity of enterocin EJ97 against B. coagulans CECT 12 Chemical preservatives used in foods as additional hurdles for preservation may act as potentiators or inhibitors of bacteriocin activity (G.anzle et al., 1998). Therefore, the activity of enterocin EJ97 was tested at sub-inhibitory concentrations (1 mg/ml) against vegetative cells of B. coagulans CECT 12 (4 105 cfu/ml) in the presence of four different chemical preservatives (Table 1). The sodium salt of benzoic acid had a slight
ARTICLE IN PRESS 536
M.T. Garc!ıa et al. / Food Microbiology 20 (2003) 533–536
inhibitory effect on bacterial growth, that was proportional to the final concentration tested. At 0.1%, sodium benzoate caused a noticeable delay on growth, but it showed no effect on cell viability. The combination of EJ97 and sodium benzoate at concentrations of 0.05% and 0.1% had a similar effect on bacterial growth as the addition of bacteriocin alone, indicating that this compound did not have any potentiating or antagonizing effect on enterocin activity. Sodium chloride had no effect on growth of B. coagulans CECT 12 at the concentrations tested (3% and 6%). Furthermore, bacteriocin activity was not modified at all by the presence of sodium chloride. The chelating agent sodium tripolyphosphate had no effect on bacterial growth at a final concentration of 0.3%. At 0.5%, some delay of growth was detected (as shown by the lower number of viable cells at 4 h of incubation in comparison with the untreated control). Sodium tripolyphosphate had no effect on the activity of enterocin EJ97 at a final concentration of 0.3%. However, when the chelating agent was tested at 0.5% in combination with EJ97, the number of viable cells was ca. one log unit lower compared to cultures treated with EJ97 alone, indicating a potentiating effect. Sodium nitrite showed a clear inhibitory effect on bacterial growth as well as cell viability, depending on the final concentration tested. After 4 h of incubation, a marked reduction in the initial concentrations of viable cells was observed for nitrite concentrations of 50 mg/ml or above. Upon incubation with sodium nitrite (50 mg/ ml) and enterocin EJ97 (1 mg/ml) viable cell counts were one log unit lower compared to cultures incubated with bacteriocin alone. The combination of enterocin EJ97 and sodium nitrite (100 mg/ml) killed all viable cells of B. coagulans CECT 12 within 4 h of incubation at 371C, indicating a synergistic action of the bacteriocin with nitrite. Activity of enterocin against L. monocytogenes is also enhanced by nitrite (unpublished results). The antimicrobial activity of other bacteriocins like enterocin AS-48 (Abriouel et al., 2002), nisin (Shahidi, 1991) or lactocin 705 (Vignolo et al., 1998) can also be enhanced by this chemical compound. Although sodium nitrite is only permitted as additive for meat products (with residual limits ranging from 50 to 175 mg/kg; European Parliament and Council Directive No. 95/2/ EC and amendments), nitrites are also found naturally in vegetable foods, and canned vegetable juices have been reported to exceed the acceptable daily intake of nitrite (Cheng and Tsang, 1998). Therefore, the influence of nitrite concentration on the activity of enterocin EJ97 in nitrite-containing foods should be studied. There are scarce data on the effects of bacteriocins against B. coagulans, although the antibiotic nisin is highly active both on vegetative cells and spores of this bacterium (Roberts and Hoover, 1996). There is a
growing interest on the combined use of bacteriocins in order to avoid selection for resistant strains. Therefore, the combined effects of nisin and enterocin EJ97 should be studied in vitro and in model food systems.
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