- Email: [email protected]
An inhibitory synergistic effect of a nisin–monolaurin combination on Bacillus sp. vegetative cells in milk
Food Microbiology, 2001, 18, 87^94 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.2000.0379
ORIGINAL ARTICLE
An inhibitory synergistic e¡ect of a nisin^monolaurin combination on Bacillus sp. vegetative cells in milk Marianne Mansour and Jean-Bernard Millie're*
The inhibitory activities of nisin and monolaurin, used alone or in combination, were investigated against four Bacillus species vegetative cells in milk at 378C for 5 days. In the absence of inhibitors, the four strains grew and sporulated at the end of the exponential growth step and throughout the stationary phase. Nisin (100 IU ml71) induced an immediate reduction in the population level but transient because regrowth appeared and was strain-dependent; cell concentrations reached the control culture level, e.g. 6^7 log(10) as well as the spore load (4^5 log(10)). On the other hand, monolaurin (250 mg ml71) had a durable bacteriostatic e¡ect followed by a regrowth level constantly lower than that of the control culture; sporulation was low (between 13 and 7610 3 spl ml71) and did not occur in the case of B. coagulans. The use of these inhibitors in combination, induced a synergistic bactericidal e¡ect leading to a total inhibition (0 cfu ml71) until day 5, except in the case of B. cereus where a concentration of 500 cfu ml71 was constant till the end of the experiment; consequently, sporulation was # 2001 Academic Press absent.
Introduction Members of the genus Bacillus are organisms widely distributed in the environment and in many food such as meat (Bell and De Lacy 1987), canned tomato (Vicini et al. 1992), spices (Logan and De Vos 1998), ¢sh sauce (Al-Jedah et al. 1999), cooked or chilled vegetables (Carlin et al. 2000) and especially dairy products (Lewis 1999). These micro-organisms act as food pathogen (B. cereus) or as spoilage bacteria (B. coagulans, B. licheniformis, B. subtilis). Bacillus species are able to form
*Corresponding author. IUT Nancy-Brabois, UHP Nancy 1 De¤partement Ge¤nie Biologique Agro-Alimentaire 54600 Villers-le's-Nancy, France. Fax: 33 3 83274205. E-mail: [email protected] 0740 -0020/01/010087 +08 $35.00/0
endospores highly resistant to many physical or chemical agents used for food preservation. The bacteriocin nisin, a biocide polypeptide produced by some strains of Lactococcus lactis is particularly e¡ective in controlling the growth of sporeformers such as Bacillus and Clostridium genera (Bell and De Lacy 1987, Oscroft et al. 1990, Roberts and Hoover 1996, Jacquette and Beuchat 1998) as well as species of Listeria monocytogenes (Budu-Amoako et al. 1999, Bouttefroy et al. 2000), Staphylococcus (Nykanen et al. 1998) and many lactic acid bacteria species (De Vuyst and Vandamme 1994). Monoglycerides are used in food industry as £avouring and emulsifying agents. These compounds, especially monolaurin, the monoester of lauric acid, have received the most attention because of its antimicrobial properties (Kabara 1993, Oh and Marshall 1995, Chaibi et al. 1996,Wang and Johnson 1997). # 2001 Academic Press
Received: 27 June 2000 ENSAIA-INPL, Laboratoire Bioproce¤de¤s AgroAlimentaires (LABIAL), 2, avenue de la Fore“t de Haye, 54505 Vandoeuvrele's-Nancy, France
88 M. Mansour and J.-B. Millie're
Generally, the use of antimicrobials alone, as a unique method of destroying spores or vegetative cells, has been opposed to some limitations especially the emergence of resistant strains (Mazzotta and Montville 1997, 1999). The antimicrobial e¡ects of nisin and monolaurin can be increased if used together or in combination with other preservative systems in order to establish a series of hurdles that micro-organisms could not overcome (Leistner and Gorris 1995). Nisin has been extensively used in combination with other treatments or antimicrobial agents such as high hydrostatic pressure (Roberts and Hoover 1996), suboptimal growth conditions (Jacquette and Beuchat 1998, Bouttefroy et al. 2000), heat treatment (Budu-Amoako et al. 1999), nitrite (Taylor et al. 1985), other bacteriocins (Hanlin et al. 1993), organic acids (Oscroft et al. 1990), potassium sorbate (Avery and Buncic 1997, Mansour et al. 1998), sucrose fatty acid esters (Thomas et al. 1998), enzymatic systems such as lactoperoxidase (Boussouel et al. 1999) and lysozyme (Padgett et al. 1998). The combination of monolaurin with heat treatment (Tsushido et al. 1981, Oh and Marshall 1995), organic acids (Bell and De Lacy 1987, Oh and Marshall 1996), other monoglycerides (Wang and Johnson 1997), and sodium citrate and eugenol (Blaszyck and Holley 1998) has been studied already. In previous work (Mansour et al. 1999), we investigated the combination of nisin and monolaurin on B. licheniformis spores in milk and showed the inhibitory synergistic e¡ect of these antimicrobials used in association. The aim of the present investigation was to assess the e¡ect of nisin and monolaurin, used alone or in combination, on the behaviour of vegetative cells of Bacillus sporulating strains belonging to four di¡erent species.
Materials and Methods
and transferred monthly on to fresh medium. Cultures were obtained by inoculating cells into IP broth (Institut Pasteur, Paris, France) at 378C for 24 h. This media consisted of (g l71): bactopeptone: 6?0; tryptone: 4?0; yeast extract: 3?0; meat extract: 1?5; glucose (Labosi, Maurepas, France): 1?0. These products were purchased from Biokar (Beauvais, France).
Inhibitors Nisin1 and monolaurin (1-monolauroyl-rac-glyce¤rol) were purchased from Sigma (Missouri, USA). Standard stock solution of nisin containing 16104 IU ml71 was prepared by dissolving 10 mg in 1 ml of 0?02 N HCl previously sterilized at 1108C for 10 min. Monolaurin stock solution prepared in ethanol (95%, Labosi) was concentrated to 50 mg ml71 (5% w/v) in order to avoid high ethanol concentration in cultures. The ¢nal concentration of ethanol used (40?6%) had no signi¢cant e¡ect on Bacillus sp. viability.
Vegetative cell and spore enumerations The initial inoculum consisted of vegetative cells grown in IP broth at 378C for 24 h. The presence of spores was checked before inoculating the skimmed milk (0% fat (w/v), Oxoid, Hampshire, UK) and throughout the experimentation period. Every sample was spread on IP agar (1?5% agar, Biokar) before and after a heat treatment at 808C for 10 min in order to evaluate total cell (TC) (before heating) and spore (Sp) counts (after heating). Plating was made after a serial decimal dilution in tryptone salt broth (g l71: tryptone: 1; sodium chloride: 8?5). Plates were incubated at 378C for 24 h and then enumerated. The di¡erence between TC and Sp allowed the estimation of vegetative cell (VC) count. Five milliliters of a 2% (w/v) agar suspension were poured as a thin overlay in order to prevent the spreading of Bacillus colonies.
Bacterial strains Four strains belonging to di¡erent Bacillus species were used: B. cereus ATCC 14579T, B. coagulans ATCC 7050T, B. subtilis ATCC 6633, and B. licheniformis ATCC 14580T. Cultures were kept on tryptose agar (Difco, Michigan, USA) at 48C
Individual or combined e¡ect of nisin and monolaurin on Bacillus sp. vegetative cells in milk The behaviour of Bacillus sp. vegetative cells (56104 cfu ml71) was investigated in skimmed
Synergistic inhibition of Bacillus sp. 89
milk in presence of nisin (100 IU ml71 ) and/or monolaurin (250 mg ml71). Experiments were carried out at 378C. Samples were removed immediately after the addition of antimicrobials and at intervals until 5 days, for TC and Sp enumerations. Before plating, samples containing nisin were incubated at 378C for 30 min in presence of 1 mg ml71 of a protease suspension (Sigma, type XIV from Streptomyces griseus) in order to denature that bacteriocin. This enzyme did not a¡ect the behaviour of Bacillus sp. vegetative cells.
Results Bacillus cereus ATCC 14579T In absence of inhibitors, B. cereus, at an inoculation level of 1?56104 cfu ml71, grew after 1 h at 378C with a speci¢c growth rate of 0?33 h71. Spores, absent in the starting inoculum, appeared after 24 h when vegetative cell population reached the end of the exponential growth phase; their number increased throughout the stationary phase and reached 2?56105 Sp ml71 after 5 days (Fig. 1). The addition of nisin (100 IU ml71) induced an immediate (55 min) reduction of 0?3 log(10) in the initial population and 1?2 log(10) after 2 h of incubation. Regrowth appeared between 2
Figure 1. Behaviour of B. cereus ATCC 14579T ve-
getative cells (1?56104 cfu ml71) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71 ) and monolaurin (250 mg ml71). Open and closed symbols represented total cell and spore counts, respectively.
and 4 h and reached the control culture level (26107 cfu ml71) after 24 h. Sporulation similar to that of the control culture, occurred after 24 h and reached a concentration of 1?56 105 Sp ml71 after 5 days. In presence of monolaurin alone (250 mg ml71), a global bacteriostatic e¡ect was noted over 5 days. A slight increase in the population level occurred at 24 h with an almost steady state till day 5 (4?16104 cfu ml71). Sporulation was lower than in the control culture with 76103 Sp ml71 after 5 days. The combination of nisin (100 IU ml71 ) and monolaurin (250 mg ml71 ) induced an immediate (55 min) bactericidal e¡ect, greater than the e¡ect of nisin alone, as indicated by the decrease in the population level of about 1?7 log(10). A bacteriostatic e¡ect (500 cfu ml71) was maintained until 5 days without regrowth and consequently without sporulation.
B. coagulans ATCC 7050T The inoculation level in milk was about 7?56104 cfu ml71 of B. coagulans vegetative cells. At the initial time (t0), spores were absent. In the absence of inhibitors, cell growth occurred after a lag time of 1 h at a speci¢c growth rate of 0?33 h71. Sporulation appeared after 24 h and spore population reached 7?56104 Sp ml71 after 5 days (Fig. 2).
Figure 2. Behaviour of B. coagulans ATCC 7050T
vegetative cells (7?56104 cfu ml71) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71) and monolaurin (250 mg ml71 ). Open and closed symbols represented total cell and spore counts, respectively.
90 M. Mansour and J.-B. Millie're
Immediately after the addition of nisin (100 IU ml71) and at 8 h incubation, the decrease in the population was about 1?9 log(10) and 3?5 log(10), respectively. This bactericidal e¡ect was maintained till 24 h with only 4 cfu ml71. Nevertheless, regrowth appeared after 48 h and after 5 days the population level was 16106 cfu ml71. Sporulation appeared at the ¢fth day of incubation. Monolaurin (250 mg ml71) exerted a bacteriostatic rather than a bactericidal e¡ect despite the reduction of 1 log(10) in the population between 0 and 8 h. After 5 days, the bacterial level, about 26105 cfu ml71 was lower than that of the control culture (26108 cfu ml71) and sporulation was not observed. Nisin and monolaurin in combination immediately induced a reduction of 3?3 log(10). This bactericidal e¡ect was stronger than that observed with nisin alone (1?9 log(10)). From 48 h to 5 days, no viable cells were detected in 1 ml of sample culture. As a result of this total inhibition, sporulation was absent.
Figure 3. Behaviour of B. subtilis ATCC 6633 ve-
getative cells (76104 cfu ml71 ) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71) and monolaurin (250 mg ml71). Open and closed symbols represented total cell and spore counts, respectively.
B. subtilis ATCC 6633 The initial cell load in milk was 76104 cfu ml71. In absence of inhibitors, B. subtilis vegetative cell growth occurred also at a speci¢c growth rate of 0?33 h71. Spores, absent in the initial inoculum, appeared after 24 h, and reached 56104 Sp ml71 after 5 days (Fig. 3). In presence of nisin (100 IU ml71), a 4 log(10) reduction was achieved in the initial population after 6 h of incubation at 378C. From 8 h, regrowth led to a population level similar to that of the control culture (56106 cfu ml71 ) after 48 h. Vegetative cells started to sporulate at 48 h and spore number was about 1?46103 Sp ml71 after 5 days. The main e¡ect of monolaurin (250 mg ml71) was bacteriostatic with an almost steady cell load (5?56104 cfu ml71) till day 5. A total of 100 Sp ml71 was detected at the ¢fth day of incubation at 378C. The simultaneous addition of nisin and monolaurin induced a bactericidal e¡ect (2 log(10) ) greater than that of nisin alone (1?5 log(10) ). This e¡ect led after 48 h of incubation to a total inhibition (absence of viable cells
Figure 4. Behaviour of B. licheniformis ATCC
14580T vegetative cells (16104 cfu ml71 ) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71 ) and monolaurin (250 mg ml71 ). Open and closed symbols represented total cell and spore counts, respectively.
in 1 ml sample culture) without regrowth until 5 days and consequently, sporulation did not occur.
B. licheniformis ATCC 14580T In absence of inhibitors, B. licheniformis, at an inoculation level of 16104 cfu ml71, grew at the same speci¢c growth rate of 0?33 h71. Spores, absent in the starting inoculum, appeared after 24 h and reached 5?26104 Sp ml71 after 5 days (Fig. 4).
Synergistic inhibition of Bacillus sp. 91
Immediately, after the addition of nisin (100 IU ml71 ) and after 8 h of incubation, the population level declined by about 1?2 log(10) and 4 log(10), respectively. Regrowth appeared after 24 h and population level reached almost that of the control culture (36107 cfu ml71) after 5 days. Sporulation occurred after 48 h with a spore number of about 3?26104 Sp ml71 after 5 days. The addition of monolaurin (250 mg ml71) induced a bacteriostatic e¡ect (16104 cfu ml71) until 8 h of incubation. Then a slight growth gave a cell load of 9?76104 cfu ml71 after 5 days. Sporulation started at the ¢fth day with a ¢nal total of 13 Sp ml71. The simultaneous addition of nisin and monolaurin exerted a bactericidal e¡ect allowing an immediate reduction of 3?5 log(10). Between 4 h and 5 days of incubation, a total inhibition was noted and consequently without the sporulation phase. The behaviour of these four Bacillus species, in matters of growth, inhibition, regrowth, and sporulation is summarized in Table 1. Actually, among the four strains tested, B. cereus was the
most resistant to the action of the inhibitors used alone or together. The other three strains had the same sensitivity to the nisin^ monolaurin combination. In presence of each inhibitor alone and despite the high inoculation level (76104cfu ml71), B. coagulans and B. subtilis were the most sensitive to nisin and monolaurin, respectively. Bacillus licheniformis (16104 cfu ml71), was less resistant than B. cereus but less sensitive than B. coagulans and B. subtilis.
Discussion This work emphasized the synergistic e¡ect of the antimicrobial activity of nisin and monolaurin, despite di¡erences in Bacillus sp. sensitivity. Although the mechanisms of action of nisin and monolaurin are di¡erent, the fact that the cytoplasmic membrane is their primary site of action could explain their inhibitory synergistic e¡ect. Nisin binds onto the membrane, inserts into it and forms pores leading to proton motive force dissipation and to
Table 1. The behaviour of Bacillus sp. in absence or presence of nisin and/or monolaurin in milk at 378C. Assays
log(10) N0
B. cereus Control Nisin Monolaurin Combination
4.18
B. coagulans Control Nisin Monolaurin Combination
4.90
B. subtilis Control Nisin Monolaurin Combination
4.80
B. licheniformis Control Nisin Monolaurin Combination
4.00
a
Immediate e¡ecta
log(10) N5d
Global e¡ectb
1.045 0.82 1.6
7.30 7.18 4.60 2.70
73.23 73.17 70.60 +1.30
^ 4.29 1.32 4.90
8.23 6.00 5.30 ^
73.33 71.10 70.40 +4.90
^ 3.85 1.40 4.80
7.00 7.00 4.74 ^
72.2 72.2 +0.06 +4.80
^ 4.00 0.30 4.00
7.30 7.50 4.98 ^
73.33 73.47 70.98 +4.00
^
: log(10) (N0 /Nmin) where N0 and Nmin represented the initial and the minimal population before regrowth, respectively; b : log(10) (N0/Nmax) where Nmax represented the population at 5 days; +: inhibitory e¡ect; 7: growth.
92 M. Mansour and J.-B. Millie're
e¥ux of many vital intracellular compounds (Abee 1995, Montville et al. 1999). The inhibitory action of monolaurin, like fatty acids in general, may be partly due to an ‘uncoupling’ e¡ect of the plasma membrane (Kabara 1993, Bracey et al. 1998, Stratford and Anslow 1998). Monolaurin a¡ects the respiratory activity of cells by the inhibition of enzymes involved in oxygen uptake (Kabara 1993) and inhibits the transport of aminoacids into cells (Galbraith and Miller 1973). The inhibition of macromolecular synthesis and cellular autolysis could also account for its antimicrobial activity (Tsushido 1994). When acting individually, nisin induced an immediate reduction in the population but transient because regrowth and sporulation rapidly occurred. Conversely, monolaurin exerted a durable bacteriostatic e¡ect followed by a weak regrowth and a sporulation phase which did not reach the control culture levels. These results are in agreement with those of Bell and De lacy (1987) and con¢rmed those of previous work aimed to study the mode of action of nisin and/or monolaurin on B. licheniformis spores in milk (Mansour et al. 1999). Generally, the ine¡ectiveness of an antimicrobial compound is related to the absence of a⁄nity between the inhibitor and the bacterial cell envelopes or to the emergence of resistant cells. This latter problem is of a major concern and many reports have suggested the failure of nisin based preservation systems to prevent the eventual growth of nisin-resistant strains (Mazzotta et al. 1997, Mazzotta and Montville 1999). Mazzotta and Montville (1999) characterized the phenotypic changes in a nisin-resistant strain of Clostridium botulinum and demonstrated a modi¢cation in the fatty acid composition of the cell membrane; these fatty acid di¡erences suggest that the membrane may be more rigid which would interfere with the pore-forming ability of nisin. Cell resistance to fatty acids has been also reported (Tsushido 1994, Holyoak et al. 1996, Mihyar et al. 1997, Stratford and Anslow 1998). Holyoak et al. (1996), who studied the adaptation and growth of Saccharomyces cerevisiae in presence of sorbic acid, reported that the yeast cell adaptation to this fatty acid is dependent on (i) the
restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy demanding process and (ii) the generation of su⁄cient ATP to drive this process and still allow growth. Such problem could be avoided by the combination of nisin and monolaurin which showed a potent inhibition of Bacillus sp. without regrowth allowing thus the safety and the extension of food product shelf-life. This work emphasized the greater e¡ect of nisin and monolaurin in combination than when each was used alone; the signi¢cance (0?0014P 50?01) of this result has been studied and reported previously (Mansour et al. 1999). Despite the fact that the hurdle concept (Leistner and Gorris 1995) is widely adopted and especially the combination of nisin or monolaurin with other preservative system, their use together has not been studied before. Only one report described a primary e¡ect of a nisin^monolaurin combination against Streptococcus agalactiae in milk at 378C (Blackburn et al. 1989). The authors found a synergistic e¡ect based on MIC assays using nisin at 2000 IU ml71 and monolaurin at 1000 mg ml71. These high concentrations are certainly e¡ective but, in the case of monolaurin, may lead to an unpleasant soapy odour and taste (Bell and De Lacy 1987) and, in the case of nisin, may exceed the maximal dose de¢ned by every country authorizing its use (Hurst and Hoover 1993). Actually, nisin is commonly used in varieties of foods including processed cheeses and other dairy products (De Vuyst and Vandamme 1994), meat products (Bell and De Lacy 1987, Beuchat et al. 1997), vegetables, liquid egg (Delves-Broughton 1990), and beverages (Ogden et al. 1988, Radler 1990). The use of monolaurin in the food industry as a preservative is still limited but it is approved as emulsi¢er in foods at levels dictated by good manufacturing practices (Kabara 1993). The synergistic e¡ect related in this study could o¡er an original alternative for controlling these ubiquitous micro-organisms and could increase the scope for nisin and monolaurin usage within the food industry.The combination of these two antimicrobials would allow a signi¢cant reduction in their e¡ective
Synergistic inhibition of Bacillus sp. 93
concentrations and would improve the shelflife of many food products.
References Abee, T. (1995) Spore-forming bacteriocins of Grampositive bacteria and self-protection mechanisms of producer organisms. FEMS Microbiol. Let. 129, 1^10. Al-Jedah, J. H., Ali, M. Z. and Robinson, R. K. (1999) Chemical and microbiological properties of Mehiawah a popular ¢sh sauce in the Gulf. J. Food Sci. Technol. 36, 561^564. Avery, S. M. and Buncic, S. (1997) Antilisterial e¡ects of a sorbate-nisin combination in vitro and on packaged beef at refrigeration temperature. J. Food Prot. 60, 1075^1080. Bell, R. G. and De Lacy, K. M. (1987) The e⁄cacy of nisin, sorbic acid and monolaurin as preservatives in pasteurised cured meat products. Food Microbiol. 4, 277^283. Beuchat, L. R., Clavero, M. R. S. and Jaquette, C. B. (1997) E¡ects of nisin and temperature on survival, growth and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy. Appl. Environ. Microbiol. 63, 1953^1958. Blackburn, P., Polack, J., Gusik, S. A. and Robino, S. D. (1989) Nisin composition for use as enhanced, broad range bactericides.WO 89/12399. Blaszyk, M. and Holley, R. A. (1998) Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms. Int. J. Food Microbiol. 39, 175^83. Boussouel, N., Mathieu, F., Benoit, V., Linder, M., Revol-Junelles, A.-M. and Millie're, J.-B. (1999) Response Surface Methodology, an approach to predict the e¡ects of lactoperoxidase system, nisin, alone or in combination, on Listeria monocytogenes in skimmed milk. J. Appl. Microbiol. 86, 642^652. Bouttefroy, A., Mansour, M., Linder, M. and Millie're, J.-B. (2000) Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach. Int. J. Food Microbiol. 54, 109^115. Bracey, D., Holyoak, C. D. and Coote, P. J. (1998) Comparison of the inhibitory e¡ect of sorbic acid and amphotericin B on Saccharomyces cerevisiae: is growth inhibition dependent on reduced intracellular pH? J. Appl. Microbiol. 85, 1056^1066. Budu-Amoako, E., Ablett, R. F., Harris, J. and Delves-Broughton, J. (1999) Combined e¡ect of nisin and moderate heat on destruction of Listeria monocytogenes in cold-pack lobster meat. J. Food Prot. 62, 46^50.
Carlin, F., Guinebretiere, M. H., Choma, C., Pasqualini, R., Braconnier, A. and Nguyen-The, C. (2000) Spore forming Bacteria in commercial cooked, pasteurized and chilled vegetable purees. Food Microbiol. 17, 153^165. Chaibi, A., Ababouch, L. H. and Busta, F. F. (1996) Inhibition of bacterial spores and vegetative cells by glycerides. J. Food Prot. 59, 716^722. Delves-Broughton, J. (1990) Nisin and its uses as food preservative. Food Technol. 44, 100^117. De Vuyst, L. and Vandamme, E. (1994) Nisin, a lantibiotic produced by Lactococcus lactis subsp. lactis: properties, biosynthesis, fermentation and applications. In Bacteriocins of Lactic acid bacteria. (Eds L. De Vuyst and E. Vandamme) pp. 151^221. Glasgow, UK, Blackie Academic and Professional Publishers. Galbraith, H. and Miller, T. B. (1973) E¡ect of long chain fatty acids on bacterial respiration and amino acid uptake. J. Appl. Bacteriol. 36, 659^675. Hanlin, M. B., Kalchayanand, N., Ray, P. and Ray, B. (1993) Bacteriocins of lactic acid bacteria in combination have greater antibacterial activity. J. Food Prot. 56, 252^255. Holyoak, C. D., Statford, M., Mc Mullin, Z., Cole, M. B., Crimmins, K., Brown, A. J. P. and Coote, P. J. (1996) Activity of the plasma membrane H+-ATPase and optimal glycolytic £ux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of weak-acid preservative sorbic acid. Appl. Environ. Microbiol. 62, 3158^3164. Hurst, A. and Hoover, D. G. (1993) Nisin. In Antimicrobials in foods. (Eds P. M. Davidson and A. H. Branen) pp. 369^395. New York, Academic Press. Jaquette, C. B. and Beuchat, L. R. (1998) Combined e¡ects of pH, nisin, and temperature on growth and survival of psychrotrophic Bacillus cereus. J. Food Prot. 61, 563^570. Kabara, J. J. (1993) Medium-chain fatty acids and esters. In Antimicrobials in foods. (Eds P. M. Davidson and A. H. Branen) pp. 307^342. New York, Academic Press. Leistner, L. and Gorris, L. G. M. (1995) Food preservation by hurdle technology. Trends Food Sci. Technol. 6, 41^46. Lewis, M. J. (1999) Microbiological issues associated with heat treated milks. Int. J. Dairy Technol. 52, 121^125. Logan, N. A. and De Vos, P. (1998) Bacillus et industrie. Bul. S.F.M. 13, 130^136. Mansour, M., Linder, M., Millie're, J.-B. and Lefebvre, G. (1998) Combined e¡ects of nisin, lactic acid and potassium sorbate on Bacillus licheniformis spores in milk. Le Lait 7, 117^128. Mansour, M., Amri, D., Bouttefroy, A., Linder, M. and Millie're, J.-B. (1999) Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations. J. Appl. Microbiol. 86, 311^324.
94 M. Mansour and J.-B. Millie're
Mazzotta, A. S. and Montville, T. (1997) Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 108C and 308C. J. Appl. Microbiol. 82, 32^38. Mazzotta, A. S. and Montville, T. (1999) Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Appl. Environ. Microbiol. 65, 659^664. Mazzotta, A. S., Crandall, A. D. and Montville, T. (1997) Nisin resistance in Clostridium botulinum spores and vegetative cells. Appl. Environ. Microbiol. 63, 2654^2659. Mihyar, G. F.,Yamani, M. I. and Al-Sa’ed, A. K. (1997) Resistance of yeast £ora of labaneh to potassium sorbate and sodium benzoate. J. Dairy Sci. 80, 2304^2309. Montville, T. J., Chung, H. J., Chikindas, M. L. and Chen, Y. (1999) Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis. Let. Appl. Microbiol. 28, 189^193. Nykanen, A.,Vesanen, S. and Kallio, H. (1998) Synergistic antimicrobial e¡ect of nisin whey permeate and lactic acid on microbes isolated from ¢sh. Let. Appl. Microbiol. 27, 345^348. Ogden, K.,Waites, M. J. and Hammond J. R. M. (1988) Nisin and brewing. J. Inst. Brew. 94, 233^238. Oh, D. H. and Marshall, D. L. (1995) Destruction of Listeria monocytogenes bio¢lms on stainless steel using monolaurin and heat. J. Food Prot. 57, 251^255. Oh, D. H. and Marshall, D. L. (1996) Monolaurin and acetic acid inactivation of Listeria monocytogenes attached to stainless steel. J. Food Prot. 59, 249^252. Oscroft, C. A., Banks, J. G. and McPhee, S. (1990) Inhibition of thermally-stressed Bacillus spores
by combinations of nisin, pH and organic acids. Lebens.Wissen. Technol. 23, 538^544. Padgett, T., Han, I. Y. and Dawson, P. L. (1998) Incorporation of food-grade antimicrobial compounds into biodegradable packaging ¢lms. J. Food Prot. 61, 1330^1335. Radler, F. (1990) Possible use of nisin in wine making. II. Experiments to control lactic acid bacteria in the production of wine. Am. J. Enol.Vitic. 41, 7^11. Roberts, C. M. and 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. Stratford, M. and Anslow, P. A. (1998) Evidence that sorbic acid does not inhibit yeast as a classic ‘weak acid preservative’. Let. Appl. Microbiol. 27, 203^206. Taylor, S. T., Somers, E. B. and Krueger, L. A. (1985) Antibotulinal e¡ectiveness of nisin-nitrite combinations in culture medium and chicken frankfurter emulsions. J. Food Prot. 48, 234^239. Thomas, L. V., Davies, E. A., Delves-Broughton, J. and Wimpenny, J. W. T. (1998) Synergist e¡ect of sucrose fatty acid esters on nisin inhibition of Gram-positive bacteria. J. Appl. Microbiol. 85, 1013^1022. Tsuchido,T. (1994) Induction of cell autolysis of Bacillus subtilis with lysophosphatidylcholine. Appl. Microbiol. Biotechnol. 41, 106^109. Tsuchido, T., Saeki T. and Shibasaki, I. (1981) Death kinetics of Escherichia coli in a combined treatment with heat and monolaurin. J. Food Safety. 3, 57^68. Vicini, E., Previdi, M. P. and Pirone, G. (1992) Ability of thermophilic bacteria to spoil tomato products. M. A. N. 10, 105^113. Wang, L. L. and Johnson, E. A. (1997) Control of Listeria monocytogenes by monoglycerides in foods. J. Food Prot. 60, 131^138.
doi:10.1006/fmic.2000.0379
ORIGINAL ARTICLE
An inhibitory synergistic e¡ect of a nisin^monolaurin combination on Bacillus sp. vegetative cells in milk Marianne Mansour and Jean-Bernard Millie're*
The inhibitory activities of nisin and monolaurin, used alone or in combination, were investigated against four Bacillus species vegetative cells in milk at 378C for 5 days. In the absence of inhibitors, the four strains grew and sporulated at the end of the exponential growth step and throughout the stationary phase. Nisin (100 IU ml71) induced an immediate reduction in the population level but transient because regrowth appeared and was strain-dependent; cell concentrations reached the control culture level, e.g. 6^7 log(10) as well as the spore load (4^5 log(10)). On the other hand, monolaurin (250 mg ml71) had a durable bacteriostatic e¡ect followed by a regrowth level constantly lower than that of the control culture; sporulation was low (between 13 and 7610 3 spl ml71) and did not occur in the case of B. coagulans. The use of these inhibitors in combination, induced a synergistic bactericidal e¡ect leading to a total inhibition (0 cfu ml71) until day 5, except in the case of B. cereus where a concentration of 500 cfu ml71 was constant till the end of the experiment; consequently, sporulation was # 2001 Academic Press absent.
Introduction Members of the genus Bacillus are organisms widely distributed in the environment and in many food such as meat (Bell and De Lacy 1987), canned tomato (Vicini et al. 1992), spices (Logan and De Vos 1998), ¢sh sauce (Al-Jedah et al. 1999), cooked or chilled vegetables (Carlin et al. 2000) and especially dairy products (Lewis 1999). These micro-organisms act as food pathogen (B. cereus) or as spoilage bacteria (B. coagulans, B. licheniformis, B. subtilis). Bacillus species are able to form
*Corresponding author. IUT Nancy-Brabois, UHP Nancy 1 De¤partement Ge¤nie Biologique Agro-Alimentaire 54600 Villers-le's-Nancy, France. Fax: 33 3 83274205. E-mail: [email protected] 0740 -0020/01/010087 +08 $35.00/0
endospores highly resistant to many physical or chemical agents used for food preservation. The bacteriocin nisin, a biocide polypeptide produced by some strains of Lactococcus lactis is particularly e¡ective in controlling the growth of sporeformers such as Bacillus and Clostridium genera (Bell and De Lacy 1987, Oscroft et al. 1990, Roberts and Hoover 1996, Jacquette and Beuchat 1998) as well as species of Listeria monocytogenes (Budu-Amoako et al. 1999, Bouttefroy et al. 2000), Staphylococcus (Nykanen et al. 1998) and many lactic acid bacteria species (De Vuyst and Vandamme 1994). Monoglycerides are used in food industry as £avouring and emulsifying agents. These compounds, especially monolaurin, the monoester of lauric acid, have received the most attention because of its antimicrobial properties (Kabara 1993, Oh and Marshall 1995, Chaibi et al. 1996,Wang and Johnson 1997). # 2001 Academic Press
Received: 27 June 2000 ENSAIA-INPL, Laboratoire Bioproce¤de¤s AgroAlimentaires (LABIAL), 2, avenue de la Fore“t de Haye, 54505 Vandoeuvrele's-Nancy, France
88 M. Mansour and J.-B. Millie're
Generally, the use of antimicrobials alone, as a unique method of destroying spores or vegetative cells, has been opposed to some limitations especially the emergence of resistant strains (Mazzotta and Montville 1997, 1999). The antimicrobial e¡ects of nisin and monolaurin can be increased if used together or in combination with other preservative systems in order to establish a series of hurdles that micro-organisms could not overcome (Leistner and Gorris 1995). Nisin has been extensively used in combination with other treatments or antimicrobial agents such as high hydrostatic pressure (Roberts and Hoover 1996), suboptimal growth conditions (Jacquette and Beuchat 1998, Bouttefroy et al. 2000), heat treatment (Budu-Amoako et al. 1999), nitrite (Taylor et al. 1985), other bacteriocins (Hanlin et al. 1993), organic acids (Oscroft et al. 1990), potassium sorbate (Avery and Buncic 1997, Mansour et al. 1998), sucrose fatty acid esters (Thomas et al. 1998), enzymatic systems such as lactoperoxidase (Boussouel et al. 1999) and lysozyme (Padgett et al. 1998). The combination of monolaurin with heat treatment (Tsushido et al. 1981, Oh and Marshall 1995), organic acids (Bell and De Lacy 1987, Oh and Marshall 1996), other monoglycerides (Wang and Johnson 1997), and sodium citrate and eugenol (Blaszyck and Holley 1998) has been studied already. In previous work (Mansour et al. 1999), we investigated the combination of nisin and monolaurin on B. licheniformis spores in milk and showed the inhibitory synergistic e¡ect of these antimicrobials used in association. The aim of the present investigation was to assess the e¡ect of nisin and monolaurin, used alone or in combination, on the behaviour of vegetative cells of Bacillus sporulating strains belonging to four di¡erent species.
Materials and Methods
and transferred monthly on to fresh medium. Cultures were obtained by inoculating cells into IP broth (Institut Pasteur, Paris, France) at 378C for 24 h. This media consisted of (g l71): bactopeptone: 6?0; tryptone: 4?0; yeast extract: 3?0; meat extract: 1?5; glucose (Labosi, Maurepas, France): 1?0. These products were purchased from Biokar (Beauvais, France).
Inhibitors Nisin1 and monolaurin (1-monolauroyl-rac-glyce¤rol) were purchased from Sigma (Missouri, USA). Standard stock solution of nisin containing 16104 IU ml71 was prepared by dissolving 10 mg in 1 ml of 0?02 N HCl previously sterilized at 1108C for 10 min. Monolaurin stock solution prepared in ethanol (95%, Labosi) was concentrated to 50 mg ml71 (5% w/v) in order to avoid high ethanol concentration in cultures. The ¢nal concentration of ethanol used (40?6%) had no signi¢cant e¡ect on Bacillus sp. viability.
Vegetative cell and spore enumerations The initial inoculum consisted of vegetative cells grown in IP broth at 378C for 24 h. The presence of spores was checked before inoculating the skimmed milk (0% fat (w/v), Oxoid, Hampshire, UK) and throughout the experimentation period. Every sample was spread on IP agar (1?5% agar, Biokar) before and after a heat treatment at 808C for 10 min in order to evaluate total cell (TC) (before heating) and spore (Sp) counts (after heating). Plating was made after a serial decimal dilution in tryptone salt broth (g l71: tryptone: 1; sodium chloride: 8?5). Plates were incubated at 378C for 24 h and then enumerated. The di¡erence between TC and Sp allowed the estimation of vegetative cell (VC) count. Five milliliters of a 2% (w/v) agar suspension were poured as a thin overlay in order to prevent the spreading of Bacillus colonies.
Bacterial strains Four strains belonging to di¡erent Bacillus species were used: B. cereus ATCC 14579T, B. coagulans ATCC 7050T, B. subtilis ATCC 6633, and B. licheniformis ATCC 14580T. Cultures were kept on tryptose agar (Difco, Michigan, USA) at 48C
Individual or combined e¡ect of nisin and monolaurin on Bacillus sp. vegetative cells in milk The behaviour of Bacillus sp. vegetative cells (56104 cfu ml71) was investigated in skimmed
Synergistic inhibition of Bacillus sp. 89
milk in presence of nisin (100 IU ml71 ) and/or monolaurin (250 mg ml71). Experiments were carried out at 378C. Samples were removed immediately after the addition of antimicrobials and at intervals until 5 days, for TC and Sp enumerations. Before plating, samples containing nisin were incubated at 378C for 30 min in presence of 1 mg ml71 of a protease suspension (Sigma, type XIV from Streptomyces griseus) in order to denature that bacteriocin. This enzyme did not a¡ect the behaviour of Bacillus sp. vegetative cells.
Results Bacillus cereus ATCC 14579T In absence of inhibitors, B. cereus, at an inoculation level of 1?56104 cfu ml71, grew after 1 h at 378C with a speci¢c growth rate of 0?33 h71. Spores, absent in the starting inoculum, appeared after 24 h when vegetative cell population reached the end of the exponential growth phase; their number increased throughout the stationary phase and reached 2?56105 Sp ml71 after 5 days (Fig. 1). The addition of nisin (100 IU ml71) induced an immediate (55 min) reduction of 0?3 log(10) in the initial population and 1?2 log(10) after 2 h of incubation. Regrowth appeared between 2
Figure 1. Behaviour of B. cereus ATCC 14579T ve-
getative cells (1?56104 cfu ml71) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71 ) and monolaurin (250 mg ml71). Open and closed symbols represented total cell and spore counts, respectively.
and 4 h and reached the control culture level (26107 cfu ml71) after 24 h. Sporulation similar to that of the control culture, occurred after 24 h and reached a concentration of 1?56 105 Sp ml71 after 5 days. In presence of monolaurin alone (250 mg ml71), a global bacteriostatic e¡ect was noted over 5 days. A slight increase in the population level occurred at 24 h with an almost steady state till day 5 (4?16104 cfu ml71). Sporulation was lower than in the control culture with 76103 Sp ml71 after 5 days. The combination of nisin (100 IU ml71 ) and monolaurin (250 mg ml71 ) induced an immediate (55 min) bactericidal e¡ect, greater than the e¡ect of nisin alone, as indicated by the decrease in the population level of about 1?7 log(10). A bacteriostatic e¡ect (500 cfu ml71) was maintained until 5 days without regrowth and consequently without sporulation.
B. coagulans ATCC 7050T The inoculation level in milk was about 7?56104 cfu ml71 of B. coagulans vegetative cells. At the initial time (t0), spores were absent. In the absence of inhibitors, cell growth occurred after a lag time of 1 h at a speci¢c growth rate of 0?33 h71. Sporulation appeared after 24 h and spore population reached 7?56104 Sp ml71 after 5 days (Fig. 2).
Figure 2. Behaviour of B. coagulans ATCC 7050T
vegetative cells (7?56104 cfu ml71) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71) and monolaurin (250 mg ml71 ). Open and closed symbols represented total cell and spore counts, respectively.
90 M. Mansour and J.-B. Millie're
Immediately after the addition of nisin (100 IU ml71) and at 8 h incubation, the decrease in the population was about 1?9 log(10) and 3?5 log(10), respectively. This bactericidal e¡ect was maintained till 24 h with only 4 cfu ml71. Nevertheless, regrowth appeared after 48 h and after 5 days the population level was 16106 cfu ml71. Sporulation appeared at the ¢fth day of incubation. Monolaurin (250 mg ml71) exerted a bacteriostatic rather than a bactericidal e¡ect despite the reduction of 1 log(10) in the population between 0 and 8 h. After 5 days, the bacterial level, about 26105 cfu ml71 was lower than that of the control culture (26108 cfu ml71) and sporulation was not observed. Nisin and monolaurin in combination immediately induced a reduction of 3?3 log(10). This bactericidal e¡ect was stronger than that observed with nisin alone (1?9 log(10)). From 48 h to 5 days, no viable cells were detected in 1 ml of sample culture. As a result of this total inhibition, sporulation was absent.
Figure 3. Behaviour of B. subtilis ATCC 6633 ve-
getative cells (76104 cfu ml71 ) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71) and monolaurin (250 mg ml71). Open and closed symbols represented total cell and spore counts, respectively.
B. subtilis ATCC 6633 The initial cell load in milk was 76104 cfu ml71. In absence of inhibitors, B. subtilis vegetative cell growth occurred also at a speci¢c growth rate of 0?33 h71. Spores, absent in the initial inoculum, appeared after 24 h, and reached 56104 Sp ml71 after 5 days (Fig. 3). In presence of nisin (100 IU ml71), a 4 log(10) reduction was achieved in the initial population after 6 h of incubation at 378C. From 8 h, regrowth led to a population level similar to that of the control culture (56106 cfu ml71 ) after 48 h. Vegetative cells started to sporulate at 48 h and spore number was about 1?46103 Sp ml71 after 5 days. The main e¡ect of monolaurin (250 mg ml71) was bacteriostatic with an almost steady cell load (5?56104 cfu ml71) till day 5. A total of 100 Sp ml71 was detected at the ¢fth day of incubation at 378C. The simultaneous addition of nisin and monolaurin induced a bactericidal e¡ect (2 log(10) ) greater than that of nisin alone (1?5 log(10) ). This e¡ect led after 48 h of incubation to a total inhibition (absence of viable cells
Figure 4. Behaviour of B. licheniformis ATCC
14580T vegetative cells (16104 cfu ml71 ) in milk at 378C. (*) in absence of inhibitors, (&) in presence of nisin (100 IU ml71), (~) in presence of monolaurin (250 mg ml71), (^) in presence of a combination of nisin (100 IU ml71 ) and monolaurin (250 mg ml71 ). Open and closed symbols represented total cell and spore counts, respectively.
in 1 ml sample culture) without regrowth until 5 days and consequently, sporulation did not occur.
B. licheniformis ATCC 14580T In absence of inhibitors, B. licheniformis, at an inoculation level of 16104 cfu ml71, grew at the same speci¢c growth rate of 0?33 h71. Spores, absent in the starting inoculum, appeared after 24 h and reached 5?26104 Sp ml71 after 5 days (Fig. 4).
Synergistic inhibition of Bacillus sp. 91
Immediately, after the addition of nisin (100 IU ml71 ) and after 8 h of incubation, the population level declined by about 1?2 log(10) and 4 log(10), respectively. Regrowth appeared after 24 h and population level reached almost that of the control culture (36107 cfu ml71) after 5 days. Sporulation occurred after 48 h with a spore number of about 3?26104 Sp ml71 after 5 days. The addition of monolaurin (250 mg ml71) induced a bacteriostatic e¡ect (16104 cfu ml71) until 8 h of incubation. Then a slight growth gave a cell load of 9?76104 cfu ml71 after 5 days. Sporulation started at the ¢fth day with a ¢nal total of 13 Sp ml71. The simultaneous addition of nisin and monolaurin exerted a bactericidal e¡ect allowing an immediate reduction of 3?5 log(10). Between 4 h and 5 days of incubation, a total inhibition was noted and consequently without the sporulation phase. The behaviour of these four Bacillus species, in matters of growth, inhibition, regrowth, and sporulation is summarized in Table 1. Actually, among the four strains tested, B. cereus was the
most resistant to the action of the inhibitors used alone or together. The other three strains had the same sensitivity to the nisin^ monolaurin combination. In presence of each inhibitor alone and despite the high inoculation level (76104cfu ml71), B. coagulans and B. subtilis were the most sensitive to nisin and monolaurin, respectively. Bacillus licheniformis (16104 cfu ml71), was less resistant than B. cereus but less sensitive than B. coagulans and B. subtilis.
Discussion This work emphasized the synergistic e¡ect of the antimicrobial activity of nisin and monolaurin, despite di¡erences in Bacillus sp. sensitivity. Although the mechanisms of action of nisin and monolaurin are di¡erent, the fact that the cytoplasmic membrane is their primary site of action could explain their inhibitory synergistic e¡ect. Nisin binds onto the membrane, inserts into it and forms pores leading to proton motive force dissipation and to
Table 1. The behaviour of Bacillus sp. in absence or presence of nisin and/or monolaurin in milk at 378C. Assays
log(10) N0
B. cereus Control Nisin Monolaurin Combination
4.18
B. coagulans Control Nisin Monolaurin Combination
4.90
B. subtilis Control Nisin Monolaurin Combination
4.80
B. licheniformis Control Nisin Monolaurin Combination
4.00
a
Immediate e¡ecta
log(10) N5d
Global e¡ectb
1.045 0.82 1.6
7.30 7.18 4.60 2.70
73.23 73.17 70.60 +1.30
^ 4.29 1.32 4.90
8.23 6.00 5.30 ^
73.33 71.10 70.40 +4.90
^ 3.85 1.40 4.80
7.00 7.00 4.74 ^
72.2 72.2 +0.06 +4.80
^ 4.00 0.30 4.00
7.30 7.50 4.98 ^
73.33 73.47 70.98 +4.00
^
: log(10) (N0 /Nmin) where N0 and Nmin represented the initial and the minimal population before regrowth, respectively; b : log(10) (N0/Nmax) where Nmax represented the population at 5 days; +: inhibitory e¡ect; 7: growth.
92 M. Mansour and J.-B. Millie're
e¥ux of many vital intracellular compounds (Abee 1995, Montville et al. 1999). The inhibitory action of monolaurin, like fatty acids in general, may be partly due to an ‘uncoupling’ e¡ect of the plasma membrane (Kabara 1993, Bracey et al. 1998, Stratford and Anslow 1998). Monolaurin a¡ects the respiratory activity of cells by the inhibition of enzymes involved in oxygen uptake (Kabara 1993) and inhibits the transport of aminoacids into cells (Galbraith and Miller 1973). The inhibition of macromolecular synthesis and cellular autolysis could also account for its antimicrobial activity (Tsushido 1994). When acting individually, nisin induced an immediate reduction in the population but transient because regrowth and sporulation rapidly occurred. Conversely, monolaurin exerted a durable bacteriostatic e¡ect followed by a weak regrowth and a sporulation phase which did not reach the control culture levels. These results are in agreement with those of Bell and De lacy (1987) and con¢rmed those of previous work aimed to study the mode of action of nisin and/or monolaurin on B. licheniformis spores in milk (Mansour et al. 1999). Generally, the ine¡ectiveness of an antimicrobial compound is related to the absence of a⁄nity between the inhibitor and the bacterial cell envelopes or to the emergence of resistant cells. This latter problem is of a major concern and many reports have suggested the failure of nisin based preservation systems to prevent the eventual growth of nisin-resistant strains (Mazzotta et al. 1997, Mazzotta and Montville 1999). Mazzotta and Montville (1999) characterized the phenotypic changes in a nisin-resistant strain of Clostridium botulinum and demonstrated a modi¢cation in the fatty acid composition of the cell membrane; these fatty acid di¡erences suggest that the membrane may be more rigid which would interfere with the pore-forming ability of nisin. Cell resistance to fatty acids has been also reported (Tsushido 1994, Holyoak et al. 1996, Mihyar et al. 1997, Stratford and Anslow 1998). Holyoak et al. (1996), who studied the adaptation and growth of Saccharomyces cerevisiae in presence of sorbic acid, reported that the yeast cell adaptation to this fatty acid is dependent on (i) the
restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy demanding process and (ii) the generation of su⁄cient ATP to drive this process and still allow growth. Such problem could be avoided by the combination of nisin and monolaurin which showed a potent inhibition of Bacillus sp. without regrowth allowing thus the safety and the extension of food product shelf-life. This work emphasized the greater e¡ect of nisin and monolaurin in combination than when each was used alone; the signi¢cance (0?0014P 50?01) of this result has been studied and reported previously (Mansour et al. 1999). Despite the fact that the hurdle concept (Leistner and Gorris 1995) is widely adopted and especially the combination of nisin or monolaurin with other preservative system, their use together has not been studied before. Only one report described a primary e¡ect of a nisin^monolaurin combination against Streptococcus agalactiae in milk at 378C (Blackburn et al. 1989). The authors found a synergistic e¡ect based on MIC assays using nisin at 2000 IU ml71 and monolaurin at 1000 mg ml71. These high concentrations are certainly e¡ective but, in the case of monolaurin, may lead to an unpleasant soapy odour and taste (Bell and De Lacy 1987) and, in the case of nisin, may exceed the maximal dose de¢ned by every country authorizing its use (Hurst and Hoover 1993). Actually, nisin is commonly used in varieties of foods including processed cheeses and other dairy products (De Vuyst and Vandamme 1994), meat products (Bell and De Lacy 1987, Beuchat et al. 1997), vegetables, liquid egg (Delves-Broughton 1990), and beverages (Ogden et al. 1988, Radler 1990). The use of monolaurin in the food industry as a preservative is still limited but it is approved as emulsi¢er in foods at levels dictated by good manufacturing practices (Kabara 1993). The synergistic e¡ect related in this study could o¡er an original alternative for controlling these ubiquitous micro-organisms and could increase the scope for nisin and monolaurin usage within the food industry.The combination of these two antimicrobials would allow a signi¢cant reduction in their e¡ective
Synergistic inhibition of Bacillus sp. 93
concentrations and would improve the shelflife of many food products.
References Abee, T. (1995) Spore-forming bacteriocins of Grampositive bacteria and self-protection mechanisms of producer organisms. FEMS Microbiol. Let. 129, 1^10. Al-Jedah, J. H., Ali, M. Z. and Robinson, R. K. (1999) Chemical and microbiological properties of Mehiawah a popular ¢sh sauce in the Gulf. J. Food Sci. Technol. 36, 561^564. Avery, S. M. and Buncic, S. (1997) Antilisterial e¡ects of a sorbate-nisin combination in vitro and on packaged beef at refrigeration temperature. J. Food Prot. 60, 1075^1080. Bell, R. G. and De Lacy, K. M. (1987) The e⁄cacy of nisin, sorbic acid and monolaurin as preservatives in pasteurised cured meat products. Food Microbiol. 4, 277^283. Beuchat, L. R., Clavero, M. R. S. and Jaquette, C. B. (1997) E¡ects of nisin and temperature on survival, growth and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy. Appl. Environ. Microbiol. 63, 1953^1958. Blackburn, P., Polack, J., Gusik, S. A. and Robino, S. D. (1989) Nisin composition for use as enhanced, broad range bactericides.WO 89/12399. Blaszyk, M. and Holley, R. A. (1998) Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms. Int. J. Food Microbiol. 39, 175^83. Boussouel, N., Mathieu, F., Benoit, V., Linder, M., Revol-Junelles, A.-M. and Millie're, J.-B. (1999) Response Surface Methodology, an approach to predict the e¡ects of lactoperoxidase system, nisin, alone or in combination, on Listeria monocytogenes in skimmed milk. J. Appl. Microbiol. 86, 642^652. Bouttefroy, A., Mansour, M., Linder, M. and Millie're, J.-B. (2000) Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach. Int. J. Food Microbiol. 54, 109^115. Bracey, D., Holyoak, C. D. and Coote, P. J. (1998) Comparison of the inhibitory e¡ect of sorbic acid and amphotericin B on Saccharomyces cerevisiae: is growth inhibition dependent on reduced intracellular pH? J. Appl. Microbiol. 85, 1056^1066. Budu-Amoako, E., Ablett, R. F., Harris, J. and Delves-Broughton, J. (1999) Combined e¡ect of nisin and moderate heat on destruction of Listeria monocytogenes in cold-pack lobster meat. J. Food Prot. 62, 46^50.
Carlin, F., Guinebretiere, M. H., Choma, C., Pasqualini, R., Braconnier, A. and Nguyen-The, C. (2000) Spore forming Bacteria in commercial cooked, pasteurized and chilled vegetable purees. Food Microbiol. 17, 153^165. Chaibi, A., Ababouch, L. H. and Busta, F. F. (1996) Inhibition of bacterial spores and vegetative cells by glycerides. J. Food Prot. 59, 716^722. Delves-Broughton, J. (1990) Nisin and its uses as food preservative. Food Technol. 44, 100^117. De Vuyst, L. and Vandamme, E. (1994) Nisin, a lantibiotic produced by Lactococcus lactis subsp. lactis: properties, biosynthesis, fermentation and applications. In Bacteriocins of Lactic acid bacteria. (Eds L. De Vuyst and E. Vandamme) pp. 151^221. Glasgow, UK, Blackie Academic and Professional Publishers. Galbraith, H. and Miller, T. B. (1973) E¡ect of long chain fatty acids on bacterial respiration and amino acid uptake. J. Appl. Bacteriol. 36, 659^675. Hanlin, M. B., Kalchayanand, N., Ray, P. and Ray, B. (1993) Bacteriocins of lactic acid bacteria in combination have greater antibacterial activity. J. Food Prot. 56, 252^255. Holyoak, C. D., Statford, M., Mc Mullin, Z., Cole, M. B., Crimmins, K., Brown, A. J. P. and Coote, P. J. (1996) Activity of the plasma membrane H+-ATPase and optimal glycolytic £ux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of weak-acid preservative sorbic acid. Appl. Environ. Microbiol. 62, 3158^3164. Hurst, A. and Hoover, D. G. (1993) Nisin. In Antimicrobials in foods. (Eds P. M. Davidson and A. H. Branen) pp. 369^395. New York, Academic Press. Jaquette, C. B. and Beuchat, L. R. (1998) Combined e¡ects of pH, nisin, and temperature on growth and survival of psychrotrophic Bacillus cereus. J. Food Prot. 61, 563^570. Kabara, J. J. (1993) Medium-chain fatty acids and esters. In Antimicrobials in foods. (Eds P. M. Davidson and A. H. Branen) pp. 307^342. New York, Academic Press. Leistner, L. and Gorris, L. G. M. (1995) Food preservation by hurdle technology. Trends Food Sci. Technol. 6, 41^46. Lewis, M. J. (1999) Microbiological issues associated with heat treated milks. Int. J. Dairy Technol. 52, 121^125. Logan, N. A. and De Vos, P. (1998) Bacillus et industrie. Bul. S.F.M. 13, 130^136. Mansour, M., Linder, M., Millie're, J.-B. and Lefebvre, G. (1998) Combined e¡ects of nisin, lactic acid and potassium sorbate on Bacillus licheniformis spores in milk. Le Lait 7, 117^128. Mansour, M., Amri, D., Bouttefroy, A., Linder, M. and Millie're, J.-B. (1999) Inhibition of Bacillus licheniformis spore growth in milk by nisin, monolaurin, and pH combinations. J. Appl. Microbiol. 86, 311^324.
94 M. Mansour and J.-B. Millie're
Mazzotta, A. S. and Montville, T. (1997) Nisin induces changes in membrane fatty acid composition of Listeria monocytogenes nisin-resistant strains at 108C and 308C. J. Appl. Microbiol. 82, 32^38. Mazzotta, A. S. and Montville, T. (1999) Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain. Appl. Environ. Microbiol. 65, 659^664. Mazzotta, A. S., Crandall, A. D. and Montville, T. (1997) Nisin resistance in Clostridium botulinum spores and vegetative cells. Appl. Environ. Microbiol. 63, 2654^2659. Mihyar, G. F.,Yamani, M. I. and Al-Sa’ed, A. K. (1997) Resistance of yeast £ora of labaneh to potassium sorbate and sodium benzoate. J. Dairy Sci. 80, 2304^2309. Montville, T. J., Chung, H. J., Chikindas, M. L. and Chen, Y. (1999) Nisin A depletes intracellular ATP and acts in bactericidal manner against Mycobacterium smegmatis. Let. Appl. Microbiol. 28, 189^193. Nykanen, A.,Vesanen, S. and Kallio, H. (1998) Synergistic antimicrobial e¡ect of nisin whey permeate and lactic acid on microbes isolated from ¢sh. Let. Appl. Microbiol. 27, 345^348. Ogden, K.,Waites, M. J. and Hammond J. R. M. (1988) Nisin and brewing. J. Inst. Brew. 94, 233^238. Oh, D. H. and Marshall, D. L. (1995) Destruction of Listeria monocytogenes bio¢lms on stainless steel using monolaurin and heat. J. Food Prot. 57, 251^255. Oh, D. H. and Marshall, D. L. (1996) Monolaurin and acetic acid inactivation of Listeria monocytogenes attached to stainless steel. J. Food Prot. 59, 249^252. Oscroft, C. A., Banks, J. G. and McPhee, S. (1990) Inhibition of thermally-stressed Bacillus spores
by combinations of nisin, pH and organic acids. Lebens.Wissen. Technol. 23, 538^544. Padgett, T., Han, I. Y. and Dawson, P. L. (1998) Incorporation of food-grade antimicrobial compounds into biodegradable packaging ¢lms. J. Food Prot. 61, 1330^1335. Radler, F. (1990) Possible use of nisin in wine making. II. Experiments to control lactic acid bacteria in the production of wine. Am. J. Enol.Vitic. 41, 7^11. Roberts, C. M. and 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. Stratford, M. and Anslow, P. A. (1998) Evidence that sorbic acid does not inhibit yeast as a classic ‘weak acid preservative’. Let. Appl. Microbiol. 27, 203^206. Taylor, S. T., Somers, E. B. and Krueger, L. A. (1985) Antibotulinal e¡ectiveness of nisin-nitrite combinations in culture medium and chicken frankfurter emulsions. J. Food Prot. 48, 234^239. Thomas, L. V., Davies, E. A., Delves-Broughton, J. and Wimpenny, J. W. T. (1998) Synergist e¡ect of sucrose fatty acid esters on nisin inhibition of Gram-positive bacteria. J. Appl. Microbiol. 85, 1013^1022. Tsuchido,T. (1994) Induction of cell autolysis of Bacillus subtilis with lysophosphatidylcholine. Appl. Microbiol. Biotechnol. 41, 106^109. Tsuchido, T., Saeki T. and Shibasaki, I. (1981) Death kinetics of Escherichia coli in a combined treatment with heat and monolaurin. J. Food Safety. 3, 57^68. Vicini, E., Previdi, M. P. and Pirone, G. (1992) Ability of thermophilic bacteria to spoil tomato products. M. A. N. 10, 105^113. Wang, L. L. and Johnson, E. A. (1997) Control of Listeria monocytogenes by monoglycerides in foods. J. Food Prot. 60, 131^138.