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Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk
International Journal of Food Microbiology 61 (2000) 169–175 www.elsevier.nl / locate / ijfoodmicro
Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk Nora Boussouel a , Florence Mathieu a , Anne-Marie Revol-Junelles a , ` a,b , * Jean-Bernard Milliere a
b
Laboratoire de Fermentations et Bioconversions Industrielles, Institut National Polytechnique de Lorraine, ´ ˆ de Haye, BP 172, Ecole Nationale Superieure d’ Agronomie et des Industries Alimentaires ( INPL-ENSAIA), 2 Avenue de la Foret F 54505 Vandoeuvre-Les-Nancy Cedex, France Institut Universitaire de Technologie ( IUT) Nancy-Brabois, Universite´ Henri Poincare´ Nancy 1, F 54600 Villers-Les-Nancy, France Received 7 June 1999; received in revised form 27 September 1999; accepted 31 May 2000
Abstract Individual or combined effects of nisin (100 or 200 IU / ml) and the lactoperoxidase system (LPS) were analysed against 1 3 10 4 cfu / ml Listeria monocytogenes ATCC 15313 cells in skim milk, at 258C for 15 days. Nisin induced an immediate bactericidal effect and LPS a 48 h bacteriostatic phase which in both cases was followed by re-growth of L. monocytogenes. LPS and nisin added together at t 0 showed a synergistic and lasting bactericidal effect which after 8 days and until 15 days resulted in no detectable cells in 1 ml of milk. When LPS was added to cells already in contact with 100 or 200 IU / ml nisin for a period of 4 h, the inhibitory activity was enhanced with no L. monocytogenes detectable after 72 or 48 h, respectively, and until 15 days. When LPS was added after 12 h, the nisin bactericidal phase was followed by re-growth. When nisin, 100 or 200 UI / ml, was added to cells already in contact with LPS over 24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively, without any re-growth. For nisin addition after 72 h, cell counts were 8 log 10 cycles lower than in the control milk after 196 h, but population levels were similar to the control within 15 days. The best combination to inhibit L. monocytogenes ATCC 15313 was nisin present at t 0 followed by the LPS addition 4 h later, when the maximum inhibitory effect of nisin was reached. 2000 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Lactoperoxidase system; Nisin; Milk; Hurdle technology
1. Introduction Listeria monocytogenes is a psychrotrophic food*Corresponding author. Tel.: 1 33-3-83-59-58-82; fax: 1 33-383-59-59-15. E-mail address: [email protected] (J.-B. ` Milliere).
borne pathogen widely distributed in the environment. This species has been detected in a variety of food and caused a number of large scale outbreaks of listeriosis in the USA, Canada, and in Europe (Farber and Peterkin, 1991). These contaminations enhance interest in antimicrobial agents for controlling L. monocytogenes growth in food industry. The lactoperoxidase–thiocyanate–hydrogen per-
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00365-2
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oxide system (LPS) is an antimicrobial system present in raw milk. In developing countries, that system significantly increases storage times of raw milk at ambient temperature (Wolfson and Sumner, 1993). Its anti-L. monocytogenes activity was reported in trypticase soy broth (TSB) and in sterile milk (Denis and Ramet, 1989; Earnshaw and Banks, 1989; Siragusa and Johnson, 1989; Bibi and Bachmann, 1990; El-Shenawy et al., 1990; Kamau et al., 1990; Gaya et al., 1991; Zapico et al., 1993). Nisin, the bacteriocin from Lactococcus lactis subsp. lactis, inhibits the growth of gram-positive food-borne pathogens or spoilage microorganisms, and is used as a natural preservative in various food processes. The sensitivity of L. monocytogenes to nisin has been demonstrated (Benkerroum and Sandine, 1988; Harris et al., 1989). This bacteriocin induces a rapid viability loss in phosphate buffer (Monticello and O’Connor, 1990), in TSBYE (Song and Richard, 1997) or in cheese (Maisnier-Patin et al., 1992), but always bacterial growth resumes after only a few hours. Hurdle technology advocates the synergistic combinations of various antibacterial techniques in order to drastically limit the growth of spoilage bacteria (Leistner and Gorris, 1995). In the production of Ricotta-type cheeses, the combination of nisin with acetic acid and sorbate controlled L. monocytogenes contamination over a long period storage (70 days) at 6–88C (Davies et al., 1997). The simultaneous use of nisin with pediocin AcH (Hanlin et al., 1993) or with leucocin F10 (Parente et al., 1998) provides a greater antibacterial activity. So, the combination of bacteriocins and any other inhibitory substances has been proposed for preventing contamination by food-borne pathogens (Muriana, 1996). Nevertheless, in raw milk, when lactic acid bacteria producing nisin were in combination with LPS at 4 or 88C, no significant reduction of L. monocytogenes counts was observed in 24 h (Rodriguez et al., 1997). However at 308C, in ultra-heat treated (UHT) milk inoculated with L. monocytogenes Ohio, simultaneous additions of LPS and nisin reduced population counts by 0.5 log 10 over a period of 24 h. The antibacterial effect was enhanced when inhibitors are added in two steps on active growing cells (Zapico et al., 1998). The strategy of addition of these both inhibitors was important, and our previous results indicated that time duration is the
predominant factor to be considered when LPS-nisin action is studied (Boussouel et al., 1999). So, the aim of this study is to compare the effects of LPS and nisin used separately or in multiple combinations on L. monocytogenes ATCC 15313 behaviour in skim milk at 258C over a long time incubation period of 15 days.
2. Materials and methods
2.1. Bacterial cultures L. monocytogenes ATCC 15313 T , the avirulent type strain obtained from the American Type Culture Collection (ATCC, Rockville, USA), was used as the target organism. This strain was selected as representative of the L. monocytogenes sensitivity, among 33 different strains, to curvaticin 13 (unpublished information), a class IIa bacteriocin produced by Lactobacillus curvatus SB13 (Sudirman et al., 1993). The stock culture was maintained at 48C in ´ slants on Trypcase-Soy Agar (TSA) (BioMerieux, Craponne, France) supplemented with 0.6% yeast extract (Biokar Diagnostics, Beauvais, France) (TSA-YE). L. monocytogenes was transferred from stock cultures into a TS-YE broth and incubated at 378C for 24 h. A second inoculation was made into reconstituted skim milk (10% w / v, Oxoid, France) which was similarly incubated.
2.2. Preparation of preservatives Stock solution of nisin (Sigma, Missouri, USA) was made at 10 mg / ml (10 4 International Unit (IU) per ml) into 0.02 mol / l HCl. After heating at 708C for 10 min, the pH value was adjusted to 6.0 with 10 mol / l NaOH. Appropriate amounts of nisin stock solution was immediately added into inoculated skim milk to give final concentrations of 100 or 200 IU per ml. The LPS was composed of Lactoperoxidase (LPO) (BioSerae, Montolieu, France); KSCN (Prolabo, Paris, France); Glucose Oxidase (GOD) (BioSerae) and glucose (Prolabo). Each component was sterilized by filtration through 0.22 mm filters (Millipore Corp., Bedford, MA, USA) and one ml of each component was added to milk exactly 5 min before inoculation. Final concentrations of LPS
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components in milk were in mg / l: LPO, 35; KSCN, 25; GOD, 1; and glucose, 200.
2.3. Bacterial counts For viable L. monocytogenes count determination, 1 ml of a given decimal dilution of the sample in Tryptone-Salt broth (TS) (Biokar Diagnostics) was plated into TSA-YE, incubated at 378C for 48 h. In order to inactivate nisin, a protease suspension (type XIV from Streptomyces griseus, Sigma, St QuentinFallavier, France) was added at a final concentration of 1 mg / ml in sample and incubated at 378C for 20 min before plating. Numerations were performed in duplicate. The absence of L. monocytogenes cells in 1 ml of milk sample was checked by an enrichment procedure in Fraser broths (Oxoid) following the AFNOR Norm (NF V08-055, AFNOR, 1993).
2.4. Effect of LPS and nisin on L. monocytogenes growth Reconstituted skim milk at 10% (w / v) was sterilized (1108C, 10 min) and the pH value was adjusted to 6.4 with sterilized (1208C, 15 min) lactic acid (99%, Prolabo). An overnight culture of L. monocytogenes was 10-fold serial diluted in TS broth and 1 ml of appropriate dilution was inoculated into 94 ml of skim milk to obtain an initial population of 10 4 colony forming units (cfu) / ml. LPS, nisin, or their combinations were added at different incubation
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times and concentrations. When LPS or nisin was omitted, an equivalent volume of sterile distilled water was added into milk. Milk without inhibitor served as a control. Each experiment was incubated at 258C and carried out in triplicate. Counts of L. monocytogenes were carried out as described above.
3. Results In skim milk control at 258C, L. monocytogenes ATCC 15313 grew rapidly from 2 3 10 4 to 2 3 10 8 cfu / ml in 20 h (Fig. 1) then the population was stable. The pH value was 6.4 throughout the 15 days of incubation. In presence of the glucose / glucoseoxidase system, cell counts were similar than those in the control. The behaviour of L. monocytogenes was then analysed at 258C in different combinations of nisin and / or LPS.
3.1. Behaviour of L. monocytogenes when LPS and nisin were added separately or combinated at t0 Addition of nisin at t 0 gave an early but a short bactericidal effect; a maximum decrease of 2 log 10 cycles was observed within 10 h with 200 IU / ml nisin, but this decrease was only 0.5 log 10 cycles with 100 IU / ml nisin (Fig. 2a). Then growth occurred to reach population levels of 10 7 or 10 8 cfu / ml within 50 h in the presence of 200 or 100 IU / ml nisin, respectively. Compared with the control assay,
Fig. 1. Behaviour of L. monocytogenes ATCC 15313 in skim milk (pH 6.4) at 258C in control milk and in the presence of glucose / glucose-oxidase system: (d) control milk without nisin or LPS; (s) glucose / glucose-oxidase system; (j) pH in control milk without nisin or LPS.
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4 h (Fig. 3a) when the population level was 5 3 10 3 cfu / ml. This addition first induced a rapid decrease of 0.6 log 10 , followed by a 24 h bacteriostatic phase, then by a continously bactericidal phase which led within 72 h to the absence of L. monocytogenes in 1 ml of milk. No re-growth was observed until 15 days. When LPS was added after 4 h to cells in contact with 200 IU / ml nisin, the population was only 9 3 10 2 cfu / ml. That combination enhanced the bactericidal effect with no detectable L. monocytogenes before 15 days. Enrichment tests confirmed this result. When LPS was added after 12 h in milk with 100 or 200 IU / ml of nisin (Fig. 3b), the cell count was 9.6 3 10 3 or 1.5 3 10 2 cfu / ml, respectively. In the assay with 100 IU / ml nisin, this LPS addition decreased the population level by 1 log 10 and the population remained constant at 10 3 cfu / ml
Fig. 2. Behaviour of L. monocytogenes ATCC 15313 in skim milk (pH 6.4) at 258C in the presence of 100 or 200 IU / ml nisin and LPS, both inhibitors were added alone or simultaneously at the beginning of the incubation time (t 0 ). (a) Nisin or LPS added alone at t 0 . (b) Nisin and LPS added simultaneously at t 0 . (a) (d) control milk without nisin or LPS; (j) 100 IU / ml nisin alone at t 0 ; (h) 200 IU / ml nisin alone added at t 0 ; (s) LPS alone at t 0 . (b) (d) control milk without nisin or LPS; (h) 100 IU / ml nisin and LPS added simultaneously at t 0 ; (j) 200 IU / ml nisin and LPS added simultaneously at t 0 .
a similar maximal population level was obtained about 30 h later. LPS added at t 0 gave a bacteriostatic phase of about 50 h. Then the re-growth phase was allowed to reach about 10 8 cfu / ml in 200 h. LPS and nisin (100 or 200 IU / ml) added simultaneously at t 0 (Fig. 2b) led to a bacteriostatic phase of about 30 h followed by a bactericidal one. At 190 h, and until 15 days, the L. monocytogenes population was less than 10 cfu / ml. Enrichment tests, performed after 120 h of incubation, indicated the absence of viable L. monocytogenes in 1 ml of milk.
3.2. Behaviour of L. monocytogenes when LPS was added on cells previously in contact with nisin over 4 or 12 h The LPS addition was made on L. monocytogenes cells previously in contact with 100 IU / ml nisin for
Fig. 3. Effect of LPS, added at 4 h (a) or at 12 h (b) in skim milk (pH 6.4) at 258C, on the behaviour of L. monocytogenes ATCC 15313 previously in contact with 100 or 200 IU / ml nisin added at t 0 . Arrow indicated time of LPS addition in milk. (a) (d) Control milk without nisin or LPS; (h) control milk with 100 IU / ml nisin alone at t 0 ; (^) control milk with 200 IU / ml nisin alone at t 0 ; (j) 100 IU / ml nisin added at t 0 and LPS added after 4 h; (m) 200 IU / ml nisin added at t 0 and LPS after 4 h. (b) (d) control milk without nisin or LPS; (h) control milk with 100 IU / ml nisin alone at t 0 ; (^) control milk with 200 IU / ml nisin alone at t 0 ; (j) 100 IU / ml nisin added at t 0 and LPS after 12 h; (m) 200 IU / ml nisin added at t 0 and LPS added after 12 h.
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until 48 h; then a slow bactericidal phase led to no detectable viable cells in 144 h, but a re-growth phase occurred and after 15 days the population level was the same as in the control. With 200 IU / ml nisin, the inhibitory effect was higher: the LPS addition induced a 2 log 10 decrease in bacterial population which remained constant at 70 cfu / ml until 96 h. No detectable L. monocytogenes level was observed at 120 h, but a re-growth phase led to population level similar as in the control milk at 15 days.
3.3. Behaviour of L. monocytogenes when nisin was added on cells previously in contact with LPS over 24 or 72 h
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(Fig. 4a). Addition of nisin at this time induced a rapid and a permanent bactericidal effect. So, L. monocytogenes was not detected at 240 h or at 196 h with 100 or 200 IU / ml nisin, respectively, even after 15 days. After 72 h of incubation with LPS, the population level reached 1 3 10 6 cfu / ml (Fig. 4b). At this time, the addition of 100 IU / ml nisin induced an immediate 4 log 10 decrease, so that after 144 h the population level was 5 log 10 cycles lower than in the assay with LPS alone. Thus, a re-growth phase started at 244 h and population level was about 1.10 8 cfu / ml after 15 days. A similar effect was obtained with 200 IU / ml nisin; the L. monocytogenes population slowed down until 1 3 10 2 cfu / ml at 196 h, then the population level increased to rise 10 8 cfu / ml after 15 days.
After 24 h of incubation at 258C with LPS, cell counts were still at the initial level of 4 10 4 cfu / ml 4. Discussion
Fig. 4. Effect of nisin, added at 24 h (a) or at 72 h (b) in skim milk (pH 6.4) at 258C on the behaviour of L. monocytogenes ATCC 15313, previously in contact with LPS added at t 0 . Arrow indicated time of nisin addition in milk. (a) (d) Control milk without nisin or LPS; (s) control milk with LPS added at t 0 ; (j) LPS added at t 0 and 100 IU / ml nisin at 24 h; (m) LPS added at t 0 and 200 IU / ml nisin at 24 h. (b) (d) Control milk without nisin nor LPS; (s) control milk with LPS added at t 0 ; (j) LPS added at t 0 and 100 IU / ml nisin at 72 h; (m) LPS added at t 0 and 200 IU / ml nisin at 72 h.
In this study, in skim milk (pH 6.4) incubated at 258C, LPS alone induced a 48 h bacteriostatic phase followed by the re-growth of the target strain L. monocytogenes ATCC 15313. These observations were in agreement with previous reports; in skim milk at 308C, LPS was essentially bacteriostatic during 8 h for L. monocytogenes and L. innocua, whereas at 208C the bacteriostatic effect was about 20 h for L. innocua (Bibi and Bachmann, 1990). At 208C in milk, LPS delayed but did not prevent the growth of L. monocytogenes Scott A (Siragusa and Johnson, 1989). In our experiments, nisin induced an immediate but transitory bactericidal effect, followed by a regrowth phase. Some studies indicated an immediate bactericidal effect after the addition of bacteriocin, with little or no effect at all after a longer time of incubation (Harris et al., 1991; Mathieu et al., 1994; Muriana, 1996). In TSBYE at 308C, 20 IU / ml nisin gave a 2 log 10 cycle reduction of L. innocua Ln11 in less than 1 h, then growth resumed (Song and Richard, 1997). In phosphate buffer, with 100 IU / ml nisin, L. monocytogenes cell numbers decreased from about 10 6 cfu / ml to about 10 3 cfu / ml in 2–3 h (Monticello and O’Connor, 1990). During camembert cheese ripening, nisin produced by Lactococcus lactis subsp. lactis, at a maximal concentration of 700 IU / ml, induced a rapid decrease in L. monocytogenes cell counts between 6 and 24 h; this inhibition continued over 2 weeks leading to a reduction of 3.3
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log 10 cycles per gram of cheese, but thereafter regrowth occurred (Maisnier-Patin et al., 1992). These behaviours may be due to the emergence of spontaneous nisin-resistant variants which are able to multiply in the presence of the bacteriocin (Harris et al., 1991; Hanlin et al., 1993; Rekhif et al., 1994; Song and Richard, 1997). Appropriate combinations of these two inhibitors which have different kinds of killing mechanisms should minimize the emergence of such resistant cells. In our study, LPS and nisin combinations enhanced the antibacterial effect. They reduced to a non detectable level an initial L. monocytogenes population of 10 4 cfu / ml and prevented the regrowth phase. When LPS and nisin were added simultaneously at t 0, an original effect was the suppression of the early and rapid bactericidal phase of nisin followed by a rapid re-growth. So, after a bacteriostatic effect, a bactericidal phase led to the reduction of cell counts by 4 log 10 cycles in 144 h and to the absence of cells until 15 days. When these two inhibitors were added in two steps, the bactericidal response was depending on the time contact between cells and the first inhibitor (LPS or nisin). LPS added 4 h after nisin led to the apparent absence of cells within 48 h. When LPS addition was after 12 h, the bactericidal phase (4 log 10 cycle reduction) was followed by re-growth after 200 h of incubation. When nisin was added to cells already in the presence of LPS over 24 h, no viable L. monocytogenes cells were detectable within 10 days, with no re-growth phase until 15 days. When the bacteriocin was added after 72 h, at a time when L. monocytogenes re-grew actively, the reduction in cell counts was 8 log 10 cycles compared with the control milk; nevertheless within 15 days population levels were similar to those of the control. The synergistic effect of LPS and nisin was in accordance with a previous study, only made over a 24 h period, which showed that LPS-nisin combination was bacteriostatic (Zapico et al., 1998). These authors observed higher antibacterial activity when the inhibitors were added in two steps on active growing cells; when LPS was added after 3 h and nisin after 5 h, a 4.5 log 10 cycles decrease in population occurred within 24 h. In our experiments, with H 2 O 2 supplied by the glucose / glucose-oxidase system, and for a long incubation time, the bacteriostatic phase was followed by a bactericidal phase which led to the apparent absence
of L. monocytogenes cells. This result confirmed that time duration is a very important facteur to consider when LPS-nisin action is studied (Boussouel et al., 1999). In our study, the second inhibitor was added after a preliminary cell contact with the first one. The bactericidal effect was more pronounced and rapid when the second addition occurred during the maximal cell viability loss induced by the first one. The better strategy of combination was LPS added 4 h after the nisin action Appropriate combinations and strategies of nisin and LPS application are able to inhibit high levels (1 3 10 4 cfu / ml) of L. monocytogenes in skim milk at 258C over a long period, 15 days. Further work is currently underway to analyse the activity of these both inhibitors in various food environments, e.g. conditions of varying pH, NaCl, etc. and at refrigeration temperatures in order to propose novel strategies to control food-borne pathogens.
Acknowledgements ´ The authors thank Delphine Maigne-Roger for her technical assistance. This work was supported by a grant from the Transfert Innovation Agro-Alimentaire-Languedoc Roussillon (TRIAL) and by Michel Degre´ from the Bioserae Society.
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Effects of combinations of lactoperoxidase system and nisin on the behaviour of Listeria monocytogenes ATCC 15313 in skim milk Nora Boussouel a , Florence Mathieu a , Anne-Marie Revol-Junelles a , ` a,b , * Jean-Bernard Milliere a
b
Laboratoire de Fermentations et Bioconversions Industrielles, Institut National Polytechnique de Lorraine, ´ ˆ de Haye, BP 172, Ecole Nationale Superieure d’ Agronomie et des Industries Alimentaires ( INPL-ENSAIA), 2 Avenue de la Foret F 54505 Vandoeuvre-Les-Nancy Cedex, France Institut Universitaire de Technologie ( IUT) Nancy-Brabois, Universite´ Henri Poincare´ Nancy 1, F 54600 Villers-Les-Nancy, France Received 7 June 1999; received in revised form 27 September 1999; accepted 31 May 2000
Abstract Individual or combined effects of nisin (100 or 200 IU / ml) and the lactoperoxidase system (LPS) were analysed against 1 3 10 4 cfu / ml Listeria monocytogenes ATCC 15313 cells in skim milk, at 258C for 15 days. Nisin induced an immediate bactericidal effect and LPS a 48 h bacteriostatic phase which in both cases was followed by re-growth of L. monocytogenes. LPS and nisin added together at t 0 showed a synergistic and lasting bactericidal effect which after 8 days and until 15 days resulted in no detectable cells in 1 ml of milk. When LPS was added to cells already in contact with 100 or 200 IU / ml nisin for a period of 4 h, the inhibitory activity was enhanced with no L. monocytogenes detectable after 72 or 48 h, respectively, and until 15 days. When LPS was added after 12 h, the nisin bactericidal phase was followed by re-growth. When nisin, 100 or 200 UI / ml, was added to cells already in contact with LPS over 24 h, L. monocytogenes was not detectable after 196 and 244 h, respectively, without any re-growth. For nisin addition after 72 h, cell counts were 8 log 10 cycles lower than in the control milk after 196 h, but population levels were similar to the control within 15 days. The best combination to inhibit L. monocytogenes ATCC 15313 was nisin present at t 0 followed by the LPS addition 4 h later, when the maximum inhibitory effect of nisin was reached. 2000 Elsevier Science B.V. All rights reserved. Keywords: Listeria monocytogenes; Lactoperoxidase system; Nisin; Milk; Hurdle technology
1. Introduction Listeria monocytogenes is a psychrotrophic food*Corresponding author. Tel.: 1 33-3-83-59-58-82; fax: 1 33-383-59-59-15. E-mail address: [email protected] (J.-B. ` Milliere).
borne pathogen widely distributed in the environment. This species has been detected in a variety of food and caused a number of large scale outbreaks of listeriosis in the USA, Canada, and in Europe (Farber and Peterkin, 1991). These contaminations enhance interest in antimicrobial agents for controlling L. monocytogenes growth in food industry. The lactoperoxidase–thiocyanate–hydrogen per-
0168-1605 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00365-2
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oxide system (LPS) is an antimicrobial system present in raw milk. In developing countries, that system significantly increases storage times of raw milk at ambient temperature (Wolfson and Sumner, 1993). Its anti-L. monocytogenes activity was reported in trypticase soy broth (TSB) and in sterile milk (Denis and Ramet, 1989; Earnshaw and Banks, 1989; Siragusa and Johnson, 1989; Bibi and Bachmann, 1990; El-Shenawy et al., 1990; Kamau et al., 1990; Gaya et al., 1991; Zapico et al., 1993). Nisin, the bacteriocin from Lactococcus lactis subsp. lactis, inhibits the growth of gram-positive food-borne pathogens or spoilage microorganisms, and is used as a natural preservative in various food processes. The sensitivity of L. monocytogenes to nisin has been demonstrated (Benkerroum and Sandine, 1988; Harris et al., 1989). This bacteriocin induces a rapid viability loss in phosphate buffer (Monticello and O’Connor, 1990), in TSBYE (Song and Richard, 1997) or in cheese (Maisnier-Patin et al., 1992), but always bacterial growth resumes after only a few hours. Hurdle technology advocates the synergistic combinations of various antibacterial techniques in order to drastically limit the growth of spoilage bacteria (Leistner and Gorris, 1995). In the production of Ricotta-type cheeses, the combination of nisin with acetic acid and sorbate controlled L. monocytogenes contamination over a long period storage (70 days) at 6–88C (Davies et al., 1997). The simultaneous use of nisin with pediocin AcH (Hanlin et al., 1993) or with leucocin F10 (Parente et al., 1998) provides a greater antibacterial activity. So, the combination of bacteriocins and any other inhibitory substances has been proposed for preventing contamination by food-borne pathogens (Muriana, 1996). Nevertheless, in raw milk, when lactic acid bacteria producing nisin were in combination with LPS at 4 or 88C, no significant reduction of L. monocytogenes counts was observed in 24 h (Rodriguez et al., 1997). However at 308C, in ultra-heat treated (UHT) milk inoculated with L. monocytogenes Ohio, simultaneous additions of LPS and nisin reduced population counts by 0.5 log 10 over a period of 24 h. The antibacterial effect was enhanced when inhibitors are added in two steps on active growing cells (Zapico et al., 1998). The strategy of addition of these both inhibitors was important, and our previous results indicated that time duration is the
predominant factor to be considered when LPS-nisin action is studied (Boussouel et al., 1999). So, the aim of this study is to compare the effects of LPS and nisin used separately or in multiple combinations on L. monocytogenes ATCC 15313 behaviour in skim milk at 258C over a long time incubation period of 15 days.
2. Materials and methods
2.1. Bacterial cultures L. monocytogenes ATCC 15313 T , the avirulent type strain obtained from the American Type Culture Collection (ATCC, Rockville, USA), was used as the target organism. This strain was selected as representative of the L. monocytogenes sensitivity, among 33 different strains, to curvaticin 13 (unpublished information), a class IIa bacteriocin produced by Lactobacillus curvatus SB13 (Sudirman et al., 1993). The stock culture was maintained at 48C in ´ slants on Trypcase-Soy Agar (TSA) (BioMerieux, Craponne, France) supplemented with 0.6% yeast extract (Biokar Diagnostics, Beauvais, France) (TSA-YE). L. monocytogenes was transferred from stock cultures into a TS-YE broth and incubated at 378C for 24 h. A second inoculation was made into reconstituted skim milk (10% w / v, Oxoid, France) which was similarly incubated.
2.2. Preparation of preservatives Stock solution of nisin (Sigma, Missouri, USA) was made at 10 mg / ml (10 4 International Unit (IU) per ml) into 0.02 mol / l HCl. After heating at 708C for 10 min, the pH value was adjusted to 6.0 with 10 mol / l NaOH. Appropriate amounts of nisin stock solution was immediately added into inoculated skim milk to give final concentrations of 100 or 200 IU per ml. The LPS was composed of Lactoperoxidase (LPO) (BioSerae, Montolieu, France); KSCN (Prolabo, Paris, France); Glucose Oxidase (GOD) (BioSerae) and glucose (Prolabo). Each component was sterilized by filtration through 0.22 mm filters (Millipore Corp., Bedford, MA, USA) and one ml of each component was added to milk exactly 5 min before inoculation. Final concentrations of LPS
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components in milk were in mg / l: LPO, 35; KSCN, 25; GOD, 1; and glucose, 200.
2.3. Bacterial counts For viable L. monocytogenes count determination, 1 ml of a given decimal dilution of the sample in Tryptone-Salt broth (TS) (Biokar Diagnostics) was plated into TSA-YE, incubated at 378C for 48 h. In order to inactivate nisin, a protease suspension (type XIV from Streptomyces griseus, Sigma, St QuentinFallavier, France) was added at a final concentration of 1 mg / ml in sample and incubated at 378C for 20 min before plating. Numerations were performed in duplicate. The absence of L. monocytogenes cells in 1 ml of milk sample was checked by an enrichment procedure in Fraser broths (Oxoid) following the AFNOR Norm (NF V08-055, AFNOR, 1993).
2.4. Effect of LPS and nisin on L. monocytogenes growth Reconstituted skim milk at 10% (w / v) was sterilized (1108C, 10 min) and the pH value was adjusted to 6.4 with sterilized (1208C, 15 min) lactic acid (99%, Prolabo). An overnight culture of L. monocytogenes was 10-fold serial diluted in TS broth and 1 ml of appropriate dilution was inoculated into 94 ml of skim milk to obtain an initial population of 10 4 colony forming units (cfu) / ml. LPS, nisin, or their combinations were added at different incubation
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times and concentrations. When LPS or nisin was omitted, an equivalent volume of sterile distilled water was added into milk. Milk without inhibitor served as a control. Each experiment was incubated at 258C and carried out in triplicate. Counts of L. monocytogenes were carried out as described above.
3. Results In skim milk control at 258C, L. monocytogenes ATCC 15313 grew rapidly from 2 3 10 4 to 2 3 10 8 cfu / ml in 20 h (Fig. 1) then the population was stable. The pH value was 6.4 throughout the 15 days of incubation. In presence of the glucose / glucoseoxidase system, cell counts were similar than those in the control. The behaviour of L. monocytogenes was then analysed at 258C in different combinations of nisin and / or LPS.
3.1. Behaviour of L. monocytogenes when LPS and nisin were added separately or combinated at t0 Addition of nisin at t 0 gave an early but a short bactericidal effect; a maximum decrease of 2 log 10 cycles was observed within 10 h with 200 IU / ml nisin, but this decrease was only 0.5 log 10 cycles with 100 IU / ml nisin (Fig. 2a). Then growth occurred to reach population levels of 10 7 or 10 8 cfu / ml within 50 h in the presence of 200 or 100 IU / ml nisin, respectively. Compared with the control assay,
Fig. 1. Behaviour of L. monocytogenes ATCC 15313 in skim milk (pH 6.4) at 258C in control milk and in the presence of glucose / glucose-oxidase system: (d) control milk without nisin or LPS; (s) glucose / glucose-oxidase system; (j) pH in control milk without nisin or LPS.
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4 h (Fig. 3a) when the population level was 5 3 10 3 cfu / ml. This addition first induced a rapid decrease of 0.6 log 10 , followed by a 24 h bacteriostatic phase, then by a continously bactericidal phase which led within 72 h to the absence of L. monocytogenes in 1 ml of milk. No re-growth was observed until 15 days. When LPS was added after 4 h to cells in contact with 200 IU / ml nisin, the population was only 9 3 10 2 cfu / ml. That combination enhanced the bactericidal effect with no detectable L. monocytogenes before 15 days. Enrichment tests confirmed this result. When LPS was added after 12 h in milk with 100 or 200 IU / ml of nisin (Fig. 3b), the cell count was 9.6 3 10 3 or 1.5 3 10 2 cfu / ml, respectively. In the assay with 100 IU / ml nisin, this LPS addition decreased the population level by 1 log 10 and the population remained constant at 10 3 cfu / ml
Fig. 2. Behaviour of L. monocytogenes ATCC 15313 in skim milk (pH 6.4) at 258C in the presence of 100 or 200 IU / ml nisin and LPS, both inhibitors were added alone or simultaneously at the beginning of the incubation time (t 0 ). (a) Nisin or LPS added alone at t 0 . (b) Nisin and LPS added simultaneously at t 0 . (a) (d) control milk without nisin or LPS; (j) 100 IU / ml nisin alone at t 0 ; (h) 200 IU / ml nisin alone added at t 0 ; (s) LPS alone at t 0 . (b) (d) control milk without nisin or LPS; (h) 100 IU / ml nisin and LPS added simultaneously at t 0 ; (j) 200 IU / ml nisin and LPS added simultaneously at t 0 .
a similar maximal population level was obtained about 30 h later. LPS added at t 0 gave a bacteriostatic phase of about 50 h. Then the re-growth phase was allowed to reach about 10 8 cfu / ml in 200 h. LPS and nisin (100 or 200 IU / ml) added simultaneously at t 0 (Fig. 2b) led to a bacteriostatic phase of about 30 h followed by a bactericidal one. At 190 h, and until 15 days, the L. monocytogenes population was less than 10 cfu / ml. Enrichment tests, performed after 120 h of incubation, indicated the absence of viable L. monocytogenes in 1 ml of milk.
3.2. Behaviour of L. monocytogenes when LPS was added on cells previously in contact with nisin over 4 or 12 h The LPS addition was made on L. monocytogenes cells previously in contact with 100 IU / ml nisin for
Fig. 3. Effect of LPS, added at 4 h (a) or at 12 h (b) in skim milk (pH 6.4) at 258C, on the behaviour of L. monocytogenes ATCC 15313 previously in contact with 100 or 200 IU / ml nisin added at t 0 . Arrow indicated time of LPS addition in milk. (a) (d) Control milk without nisin or LPS; (h) control milk with 100 IU / ml nisin alone at t 0 ; (^) control milk with 200 IU / ml nisin alone at t 0 ; (j) 100 IU / ml nisin added at t 0 and LPS added after 4 h; (m) 200 IU / ml nisin added at t 0 and LPS after 4 h. (b) (d) control milk without nisin or LPS; (h) control milk with 100 IU / ml nisin alone at t 0 ; (^) control milk with 200 IU / ml nisin alone at t 0 ; (j) 100 IU / ml nisin added at t 0 and LPS after 12 h; (m) 200 IU / ml nisin added at t 0 and LPS added after 12 h.
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until 48 h; then a slow bactericidal phase led to no detectable viable cells in 144 h, but a re-growth phase occurred and after 15 days the population level was the same as in the control. With 200 IU / ml nisin, the inhibitory effect was higher: the LPS addition induced a 2 log 10 decrease in bacterial population which remained constant at 70 cfu / ml until 96 h. No detectable L. monocytogenes level was observed at 120 h, but a re-growth phase led to population level similar as in the control milk at 15 days.
3.3. Behaviour of L. monocytogenes when nisin was added on cells previously in contact with LPS over 24 or 72 h
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(Fig. 4a). Addition of nisin at this time induced a rapid and a permanent bactericidal effect. So, L. monocytogenes was not detected at 240 h or at 196 h with 100 or 200 IU / ml nisin, respectively, even after 15 days. After 72 h of incubation with LPS, the population level reached 1 3 10 6 cfu / ml (Fig. 4b). At this time, the addition of 100 IU / ml nisin induced an immediate 4 log 10 decrease, so that after 144 h the population level was 5 log 10 cycles lower than in the assay with LPS alone. Thus, a re-growth phase started at 244 h and population level was about 1.10 8 cfu / ml after 15 days. A similar effect was obtained with 200 IU / ml nisin; the L. monocytogenes population slowed down until 1 3 10 2 cfu / ml at 196 h, then the population level increased to rise 10 8 cfu / ml after 15 days.
After 24 h of incubation at 258C with LPS, cell counts were still at the initial level of 4 10 4 cfu / ml 4. Discussion
Fig. 4. Effect of nisin, added at 24 h (a) or at 72 h (b) in skim milk (pH 6.4) at 258C on the behaviour of L. monocytogenes ATCC 15313, previously in contact with LPS added at t 0 . Arrow indicated time of nisin addition in milk. (a) (d) Control milk without nisin or LPS; (s) control milk with LPS added at t 0 ; (j) LPS added at t 0 and 100 IU / ml nisin at 24 h; (m) LPS added at t 0 and 200 IU / ml nisin at 24 h. (b) (d) Control milk without nisin nor LPS; (s) control milk with LPS added at t 0 ; (j) LPS added at t 0 and 100 IU / ml nisin at 72 h; (m) LPS added at t 0 and 200 IU / ml nisin at 72 h.
In this study, in skim milk (pH 6.4) incubated at 258C, LPS alone induced a 48 h bacteriostatic phase followed by the re-growth of the target strain L. monocytogenes ATCC 15313. These observations were in agreement with previous reports; in skim milk at 308C, LPS was essentially bacteriostatic during 8 h for L. monocytogenes and L. innocua, whereas at 208C the bacteriostatic effect was about 20 h for L. innocua (Bibi and Bachmann, 1990). At 208C in milk, LPS delayed but did not prevent the growth of L. monocytogenes Scott A (Siragusa and Johnson, 1989). In our experiments, nisin induced an immediate but transitory bactericidal effect, followed by a regrowth phase. Some studies indicated an immediate bactericidal effect after the addition of bacteriocin, with little or no effect at all after a longer time of incubation (Harris et al., 1991; Mathieu et al., 1994; Muriana, 1996). In TSBYE at 308C, 20 IU / ml nisin gave a 2 log 10 cycle reduction of L. innocua Ln11 in less than 1 h, then growth resumed (Song and Richard, 1997). In phosphate buffer, with 100 IU / ml nisin, L. monocytogenes cell numbers decreased from about 10 6 cfu / ml to about 10 3 cfu / ml in 2–3 h (Monticello and O’Connor, 1990). During camembert cheese ripening, nisin produced by Lactococcus lactis subsp. lactis, at a maximal concentration of 700 IU / ml, induced a rapid decrease in L. monocytogenes cell counts between 6 and 24 h; this inhibition continued over 2 weeks leading to a reduction of 3.3
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log 10 cycles per gram of cheese, but thereafter regrowth occurred (Maisnier-Patin et al., 1992). These behaviours may be due to the emergence of spontaneous nisin-resistant variants which are able to multiply in the presence of the bacteriocin (Harris et al., 1991; Hanlin et al., 1993; Rekhif et al., 1994; Song and Richard, 1997). Appropriate combinations of these two inhibitors which have different kinds of killing mechanisms should minimize the emergence of such resistant cells. In our study, LPS and nisin combinations enhanced the antibacterial effect. They reduced to a non detectable level an initial L. monocytogenes population of 10 4 cfu / ml and prevented the regrowth phase. When LPS and nisin were added simultaneously at t 0, an original effect was the suppression of the early and rapid bactericidal phase of nisin followed by a rapid re-growth. So, after a bacteriostatic effect, a bactericidal phase led to the reduction of cell counts by 4 log 10 cycles in 144 h and to the absence of cells until 15 days. When these two inhibitors were added in two steps, the bactericidal response was depending on the time contact between cells and the first inhibitor (LPS or nisin). LPS added 4 h after nisin led to the apparent absence of cells within 48 h. When LPS addition was after 12 h, the bactericidal phase (4 log 10 cycle reduction) was followed by re-growth after 200 h of incubation. When nisin was added to cells already in the presence of LPS over 24 h, no viable L. monocytogenes cells were detectable within 10 days, with no re-growth phase until 15 days. When the bacteriocin was added after 72 h, at a time when L. monocytogenes re-grew actively, the reduction in cell counts was 8 log 10 cycles compared with the control milk; nevertheless within 15 days population levels were similar to those of the control. The synergistic effect of LPS and nisin was in accordance with a previous study, only made over a 24 h period, which showed that LPS-nisin combination was bacteriostatic (Zapico et al., 1998). These authors observed higher antibacterial activity when the inhibitors were added in two steps on active growing cells; when LPS was added after 3 h and nisin after 5 h, a 4.5 log 10 cycles decrease in population occurred within 24 h. In our experiments, with H 2 O 2 supplied by the glucose / glucose-oxidase system, and for a long incubation time, the bacteriostatic phase was followed by a bactericidal phase which led to the apparent absence
of L. monocytogenes cells. This result confirmed that time duration is a very important facteur to consider when LPS-nisin action is studied (Boussouel et al., 1999). In our study, the second inhibitor was added after a preliminary cell contact with the first one. The bactericidal effect was more pronounced and rapid when the second addition occurred during the maximal cell viability loss induced by the first one. The better strategy of combination was LPS added 4 h after the nisin action Appropriate combinations and strategies of nisin and LPS application are able to inhibit high levels (1 3 10 4 cfu / ml) of L. monocytogenes in skim milk at 258C over a long period, 15 days. Further work is currently underway to analyse the activity of these both inhibitors in various food environments, e.g. conditions of varying pH, NaCl, etc. and at refrigeration temperatures in order to propose novel strategies to control food-borne pathogens.
Acknowledgements ´ The authors thank Delphine Maigne-Roger for her technical assistance. This work was supported by a grant from the Transfert Innovation Agro-Alimentaire-Languedoc Roussillon (TRIAL) and by Michel Degre´ from the Bioserae Society.
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