Inhibition of Listeria monocytogenes by Lactobacillus sake strains of meat origin

Meat Science 38 (1994) 17-26 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0309-1740/94/$07.00

ELSEVIER

Inhibition of Listeria monocytogenes by Lactobacillus sake Strains of M e a t Origin

J. M. Rodriguez, O. J. Sobrino, W. L. Moreira, M. F. Fern~indez, L. M. Cintas, P. Casaus, B. Sanz & P. E. Hern~mdez Departarnento de Nutrici6n y Bromatologia III, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain (Received 15 July 1993; accepted 26 August 1993)

ABSTRA CT The ability o f two Lactobacillus sake strains o f meat origin to &hibit the growth o f Listeria monocytogenes at 4, 8, 15, 24 and 32°C in a conventional liquid media was investigated. Growth o f L. monocytogenes was affected by Lac. sake strains at all temperatures. The inhibition was higher at 15, 24 and 32°C than at refrigeration temperatures. The inhibitory activity o f both lactobacilli was similar perhaps due to the fact that Lac. sake 148 produces a bacteriocin inhibitory to L. monocytogenes, while Lac. sake 23 is a strong lactic acid producer. The antagonism exhibited by the lactobacilli on the L. monocytogenes strains seems to display a bacteriostatic rather than a bacteriocidal effect.

INTRODUCTION The realisation that foodborne transmission of Lister& monocytogenes plays an important role in the aetiology o f human listeriosis has caused great concern to the food industry. The clinical syndromes associated with listeriosis range from an illness similar to mild influenza to severe conditions including meningo-encephalitis, septicaemia, abortion and endocarditis (Lovett, 1989; Farber & Peterkin, 1991). Listeriosis occurs especially within certain predisposed segments of the population: as pregnant women and their foetuses, newborn children, the elderly and inmunocompromised individuals. In such patients, listeriosis is associated with a high mortality rate, ranging from 13 to 34% in typical foodborne outbreaks (Rocourt, 1991). Due to this high fatality rate, listeriosis is regarded to cost much more per case and to attract more public concern than other more common foodborne diseases. In fact, listeriosis seems to be the leading fatal food17

J. M. Rodriguez et al.

18

borne infection in the US (Gellin et al., 1991), being responsible for 13% of the estimated annual numbers of deaths caused by foodborne diseases (Todd, 1989), Meat and meat products are frequently contaminated with L. monocytogenes (Grau & Vanderlinde, 1992; Hudson et al., 1992). These bacteria constitute a special threat to public health because of their ability to survive and grow at refrigeration temperatures and under other adverse conditions (Farrag & Marth, 1989). Reports of sporadic listeric infections following the consumption of contaminated cooked chilled chicken (Kerr et al., 1988) and turkey frankfurters (Barnes et al., 1989) have provided direct evidences of listeriosis linked to meat and meat products. The microbial safety of many food products could be strengthened with antimicrobial agents added to or formed in them. Many lactic acid bacteria have the ability to preserve meat and meat products by lowering their pH through the production of organic acids. Moreover, some of these bacteria produce, besides organic acids, bacteriocins with the ability to inhibit the growth of L. monoeytogenes (Schillinger & LOcke, 1989; Motlagh et al., 1992). The purpose of this study was to evaluate the inhibitory activity of two Lactobacillus sake strains from meat origin, including the bacteriocinogenic strain Lac. sake 148 (Rodriguez et al., 1989; Sobrino et al., 1991, 1992), on three L. monocytogenes strains considered pathogenic in humans

MATERIAL AND METHODS

Microorganisms The lactobacilli were previously isolated from Spanish dry fermented sausages, being selected from 720 isolates on the basis of their antagonistic activity to a number of selected indicator microorganisms. Two of the isolates were further identified as Lactobacillus sake according to the identification scheme of Schillinger and LOcke (1987), being recorded as Lac. sake 23 and Lac. sake 148 (Rodriguez et al, 1989; Sobrino et al., 1991). The three strains of L. monocytogenes employed in this study were L. monocytogenes NCTC7973, L. monocytogenes Scott A and L. monocytogenes LI1 sv4, all of them kindly provided by the Unidad de Microbiologia, Dpto. Patologia Animal I, Facultad de Veterinaria, Madrid, Spain.

Preparation of mixed cultures Mixed cultures were prepared in flasks containing 200 ml of Bacto APT broth (Difco, Detroit, MI, USA).. Accordingly, Lac. sake 23 and Lac. sake 148 were independently cultured in mixed cultures with L. monocytogenes NCTC7973, L. monocytogenes LI1 sv4 and L. monocytogenes Scott A. Pure cultures of each microorganism were also prepared. The starting inoculum of the L. monocytogenes and Lac. sake strains were always adjusted to 1 × 103 and 1 × 105 CFU/ml, respectively. The cultures were incubated at 4, 8, 15, 24 and 32°C until the lactobacilli reached their stationary phase of growth.

Inhibition ofListeria monocytogenes by Lactobacillus sake

19

Enumeration of L. monocytogenes and Lac. sake strains At each sampling interval, aliquots containing 10 ml of the cultures were removed from the flasks. L. monoeytogenes strains were counted by plating suitably diluted samples onto plates o f Listeria Selective Agar Modified Medium (LSAMM), prepared as previously described by Blanco et al. (1989). Lae. sake counts were determined by surface plating of suitably diluted samples onto MRS (De Man et al., 1960) broth, (Oxoid Ltd. Basingstoke, U K ) plates. The L S A M M and MRS plates were incubated at 37 °C for 72 h and at 32°C for 48 h, respectively. pH and lactic acid determinations The p H of the samples was determined by using a Crison pH meter (Digit 501). The concentration of L(+)-lactic acid in the cultures were determined by an enzymatic assay with a commercial kit (Boehringer Mannheim, Mannheim, Germany). Measurement of the bacteriocin activity The supernatants to measure the bacteriocinogenic activity of Lac. sake 148 were obtained from the fractions o f the cultures removed at each sampling time. The corresponding cell-free solutions were obtained by centrifuging the cultures at 12000 x g for 10 min. This was followed by neutralization of the supernatants to p H 6.1 with 1 M N a O H and filtration through a 0-22 /xm pore size filters (Millipore, Bedford, MA, USA). F o r measurement of their antimicrobial (bacteriocin) activity, an agar diffusion test was performed as described previously (Sobrino et al., 1991). RESULTS Results obtained (Figs 1 and 2) indicate that when the listeriae grew alone in the A P T broth, all of them increased their populations from the initial 1 x 103 CFU/ml to above 1 x 10 9 CFU/ml at all temperatures. However, the results shown in Fig. 1 also indicate that in the presence o f Lactobacillus sake 23, the maximum populations reached by Listeria monoeytogenes NCTC7973 were around 6.5 X 105 CFU/ml at 4 and 8°C, and between 3 and 5 x l0 s CFU/ml at 15, 24 and 32°C. Subsequently, the L. monoeytogenes NCTC7973 counts decreased to around 0-6 log lower at 4 and 8°C and 1-5 log lower at the other temperatures. The other L. monoeytogenes strains (Scott A and LI1 sv4) reached higher final populations while their behaviour in the mixed cultures was similar. Both strains attained maximum populations of around 1 x 10 7 CFU/ml at 4°C, 3 x 106 CFU/ml at 8°C and 1 x 10 6 CFU/ml at 15, 24 and 32°C, while after that, their counts remained constant or decreased very slightly until the end of the incubation. The final counts reached by Lac. sake 23 in both mixed and pure cultures were always around 1 x 109 CFU/ml at all experimental temperatures evaluated (results not shown). The results from Fig. 2 indicate that in the presence o f Lac. sake 148 the maximum and final populations reached by the L. monoeytogenes strains were simi-

20

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Fig. 1. Growth of Listeria monoeytogenes alone and in the presence of Lactobacillus sake 23 in APT broth at (A) 4°C, (B) 8°C, (C) 15°C, (D) 24°C and (E) 32°C.

lar to the ones reached in the presence of Lac. sake 23. Similar to Lac. sake 23, the final populations reached by Lac. sake 148, at all temperatures and cultures (results not shown), were always around 1 x 109 CFU/ml. Figure 3 shows the production of L(+)-lactic acid in the mixed cultures of the lactobacilli with the listeriae strains at 4, 8, 15, 24 and 32°C. It seems that a close relationship exists between inhibition of L. monocytogenes strains and the lactic acid produced by the lactobacilli. By comparing Figs 1, 2 and 3, it should be noted that L. monoeytogenes counts were stabilised or started to decrease, at all temperatures tested just when the concentration of L(+) lactic acid began to be detectable in the culture medium.

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Fig. 2. Growth of Listeria monocytogenes alone and in the presence of Lactobacillus sake 148 in APT broth at (A) 4°C, (B) 8°C, (C) 15°C, (D) 24°C and (E) 32°C.

Table 1 shows the final pH and L(+)-lactic acid concentrations of the mixed cultures after their incubations at 4, 8, 15, 24 and 32°C. Although Fig. 3 and Table 1 reveal that Lac. sake 23 is a stronger lactic acid producer than Lac. sake 148, a bacteriocinogenic strain, it has been already shown that the inhibitory activities of both lactobacilli on the L. monocytogenes strains are similar. Figure 4 shows that the antimicrobial (bacteriocin) activity of Lac. sake 148 was detectable in all cultures at the end of their exponential phase of growth and during the stationary phase. The bacteriocin production was higher as the incubation temperature increased.

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Fig. 3. Final pH and L(+)-lactic acid concentration of the mixed cultures of Lactobacillus sake and Listeria monoeytogenes strains grown in APT broth at (A) 4°C, (B) 8°C, (C) 15°C, (D) 24°C and (E) 32°C. DISCUSSION The bacteriostatic and, in some cases, bactericidal effect of lactic acid bacteria on the growth of Listeria monocytogenes growth has been described (Raccach et al., 1989; Yousef et al., 1991). Meanwhile, Buchanan et al. (1989), Johnson et al. (1988) and Shelef (1989) have shown the survival but not growth of L. monocytogenes in raw meat, ground beef and poultry products. The presence of lactic acid bacteria with bacteriostatic activity in meat and meat products may play an important role in this phenomenon. Leistner et al. (1989) have also indicated

Inhibition of Listeria monocytogenes by Lactobacillus sake

23

TABLE 1

Final pH and L(+)-Lactic Acid Concentration of Mixed Cultures of Lactobacillus sake and Listeria monocytogenes Strains Grown at Various Temperatures Mixed culture~

Temperature 4

8

15

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32

pH

Lb

pH

L

pH

L

L

23-N 23-S 23-L

4-82 4-65 4.62

7.6 7.7 7-9

4-53 4-51 4.50

8.4 8.6 8-7

4.49 4.47 4.45

9-5 9.7 9.8

4.45 10.8 4.45 10-9 4.43 11.2

4.38 11-6 4.38 12.0 4-37 12.1

148-N 148-S 148-L

4.93 4.91 4.84

6.5 6-6 6-7

4.79 4.73 4.70

7.0 7.1 7.2

4.53 4.52 4.52

7.9 8.0 8-0

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4-45 4.44 4.42

8-4 8-6 8.7

pH

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9.5 9-5 9.6

a Strain abbreviations: (23), Lac. sake 23; (148), Lac. sake 148; (N), L. monoeytogenes NCT7973; (S), L. monocytogenes Scott A; (L), L. monoeytogenes LI1 sv4. bE refers to L(+)-lactic acid (mg/ml). that although L. monocytogenes grows in sterile meat, this organism is unable to grow in the presence of a background flora essentially composed by lactobacilli. It is also well known that during manufacture and storage of fermented sausages there is a notable reduction in the numbers of viable listeriae (Grau & Vanderline, 1992). In this work, the authors have evaluated the ability o f two Lactobacillus sake strains from meat origin to inhibit the growth o f pathogenic L. monoeytogenes strains in mixed cultures in APT broth. This model system for evaluation of inhibition of listeriae strains could generate results of interest, since it has been already reported that Lac. sake are the predominant lactic acid bacteria in dry fermented sausages (L(icke, 1986). Besides, both lactobacilli are able to grow at 4°C, being potentially useful to inhibit the growth of foodborne pathogens in refrigerated meat and meat products (Sobrino et al., 1991). Finally, one strain is a higher lactic acid producer while the other has a bacteriocinogenic activity, thus providing the possibility of comparing their antagonistic activities against the same target organisms. In this study both lactobacilli prevented the L. monocytogenes strains from attaining the numbers they reached in pure cultures at all the temperatures evaluated. The lactic acid produced by the lactobacilli seems to be a major factor contributing to the inhibition of L. monocytogenes, a microorganism not particularly resistant to organic acids (Johnson et al., 1990). When lactic acid is employed as acidulant in Brain Heart Infusion (BHI) broth (Oxoid) the minimum p H for L. monoeytogenes growth ranges from 5.0 to 5.5 at 4°C, and from 4.9 to 5.1 at 30°C (Farber et al, 1989). Similarly, Glass and Doyle (1989) have shown that the growth of this organism on meat is highly dependant on pH, being unable to grow on meats near or below p H 5.0. It is likely that high concentrations o f organic acids induce severe injuries, or even the death, to L. monocytogenes cells (E1-Shenawy & Marth, 1992). The role of acidity in L. monoeytogenes inhibition is reinforced by the fact that consumption of antiacids

24

J . M . Rodriguez et al.

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Fig. 4. Bacteriocin (Sakacin M) production by Lactobacillus sake 148 growing in mixed cultures with Listeria monocytogenes strains in APT broth at (A) 4°C, (B) 8°C, (C) 15°C, (D) 24°C and (E) 32°C. by patients suffering gastric disorders is a factor predisposing them to foodborne listeriosis (Farber, 1989). However, a recent study (Kroll & Patchett, 1992) suggests that growth of L. monocytogenes in foods with a moderately low pH can notably increase their resistance in lower pH foods. The inhibition caused by both Lac. sake strains on the L. monocytogenes, strains was similar despite the fact that Lac. sake 23 is a stronger lactic acid producer than Lac. sake 148. This result is not surprising since although Lac. sake 148 is a lower lactic acid producer it also produces sakacin M (Sobrino et al., 1992), a bacteriocin like substance inhibitory to L. monocytogenes (Sobrino

Inhibition of Listeria monocytogenes by Lactobacillus sake

25

et al., 1991). Several studies have previously demonstrated inhibition of L. monocytogenes by bacteriocin-producing strains of Lactobacillus (Schillinger et al., 1991), Laetococeus (Motlagh et al., 1991), Pedioeoecus (Foegeding et al., 1992) and Leueonostoc (Hastings & Stiles, 1991). However, L. monoeytogenes strains differ greatly in their sensitivity to a particular bacteriocin, and there also seems to exist a varying sensitivity among cells of the same strain (Motlagh et al, 1991). Accordingly, the existance of L. monoeytogenes bacteriocin-resistant mutants should be expected when a bacteriocin is employed as an antimicrobial agent in foods (Harris et al., 1991; Motlagh et al., 1992). To prevent this phenomen, the use of bacterial cultures producing several bacteriocins should be considered. The continuous advances in the knowledge of the genetics of the lactic acid bacteria may provide the means to reach this objective. L. monoeytogenes is an organism widely distributed in the environment and withstands a large variety of adverse factors including high NaC1 concentration, relatively low p H levels and refrigeration temperatures. According to Shahamat et al. (1980) and Buchanan et al. (1989), lactic acid bacteria and/or their bacteriocins could be used in combination with other antimicrobial factors and good manufacturing practices to ensure the microbial safety of meat and meat products. It is known that the sensitivity of L. monocytogenes to nisin is greatly enhanced if the culture medium contains 2% NaC1 or if its pH is reduced with lactic acid (Harris et al, 1991). In this study, it has been shown that strains of Lac. sake are effective to inhibit the growth of L. monocytogenes in mixed cultures at various temperatures. Lactic acid bacteria could contribute, along with other factors, to avoid L. monocytogenes health hazards in foods. The meat origin of the authors' lactobacilli together with the bacteriocinogenic ability of Lae. sake 148 make them attractive for their use as safety factors in meat and meat products; however, further studies are necessary to determine their effectiveness in these foods.

ACKNOWLEDGEMENTS This work is supported by grant no ALI91-0255 from the Comisi6n Interministerial de Ciencia y Tecnologia (CICYT), Spain. The authors express their appreciation to the Unidad de Microbiologia, Dpto. Patologia Animal 1, Facultad de Veterinaria, Madrid (Spain), for its generous gift of the L. monocytogenes strains. J.M.R. and L.M.C. are recipients of Fellowships from the Ministerio de Educaci6n y Ciencia, Spain; W.L.M. holds a Fellowship from the CNPq, Brasil.

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