Use of enterocin CCM 4231 to control Listeria monocytogenes in experimentally contaminated dry fermented Hornád salami

International Journal of Food Microbiology 52 (1999) 115–119 www.elsevier.nl / locate / ijfoodmicro

Short communication

Use of enterocin CCM 4231 to control Listeria monocytogenes in ´ salami experimentally contaminated dry fermented Hornad A. Laukova´ a , *, S. Czikkova´ a , S. Laczkova´ b , P. Turek b b

a ˇ ´ ˇ , Slovakia Institute of Animal Physiology, Slovak Academy of Sciences, Soltesovej 4 -6, 04001 Kosice ´ 73, 04181 Kosice ˇ , Slovakia Department of Food Hygiene and Technology, University of Veterinary Medicine, Komenskeho

Received 27 October 1998; received in revised form 6 May 1999; accepted 5 August 1999

Abstract ´ The effectiveness of enterocin CCM 4231 in controlling Listeria monocytogenes contamination in dry fermented Hornad salami was examined. Three independent salami treatments were conducted under pilot plant and laboratory conditions. Salamis were produced according to standard technological parameters and stages with ripening for 3 weeks. The reference samples consisted of the meat mixture without either L. monocytogenes or bacteriocin addition. The control sample (CS) consisted of the meat mixture with 1% of L. monocytogenes inoculum (10 8 cfu ml 21 ) added; while the experimental sample (ES) consisted of the same mixture with enterocin CCM 4231 (12 800 AU g 21 ) added. Sampling was done on the first day of the experiment, before and after bacteriocin addition for ES, on the second day and after 1, 2 and 3 weeks. The enterocin addition resulted in the reduction of L. monocytogenes by 1.67 log cycle in the ES when compared to the CS immediately after addition of the bacteriocin. Although on the second day, the growth of L. monocytogenes in ES reached 3.38 cfu g 21 (log 10), a difference of 1.72 log was found between the ES and the CS. After 1 week of ripening, the L. monocytogenes count in the CS reached 10 7 cfu g 21 ; while in the ES the count was 10 4 cfu g 21 , a difference which was maintained after 2 and 3 weeks of ripening. However, bacteriocin activity in the ES could not be detected analytically. The meat mixture used did not contain Listeria.  1999 Elsevier Science B.V. All rights reserved. Keywords: Enterocin; L. monocytogenes; Dry-fermented salami

1. Introduction Listeria spp. are distributed worldwide and they have been isolated from a very wide range of sources (Farber and Peterkin, 1991). Listeria monocytogenes *Corresponding author. Tel.: 1 421-95-633-6251; fax: 1 42195-762-162. ´ E-mail address: [email protected] (A. Laukova)

comprises a group of pathogens which are probably not host adapted and occur widely in the environment as many different types. For humans, the majority of infection probably results from ingestion of food containing large numbers of the bacterium. Therefore, a huge interest in food-borne listeriosis is noted (McLauchlin, 1997). Within the last decade, biopreservation has received increased attention as a new preservation method to control pathogenic and

0168-1605 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00125-7

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spoilage bacteria in foods. Biopreservation includes the use of bacteriocin producing lactic acid bacteria (LAB) or addition of their bacteriocins. Both possibilities have been shown effective against Listeria spp. (Ben Embarek et al., 1994; Nilsson et al., 1997). Enterococci are widespread organisms which can be encountered in different environments, including foods (Tarelli et al., 1994; Casaus et al., 1997; Waite et al., 1998). They also produce bacteriocins with predominant antilisterial effect (Giraffa et al., 1995; Maisner-Patin et al., 1996; Nunez et al., 1997). The bacteriocin enterocin CCM 4231 is a small, thermostable, hydrophobic substance with a broad antimicrobial spectrum against Gram-positive as well as against some of Gram-negative organisms which is produced by Enterococcus faecium CCM 4231 (Laukova´ et al., 1997). The successful use of bacteriocins to inhibit pathogens in the experimental sausage manufacture has already been reported (Winkowski et al., 1993; Samelis et al., 1994). The aim of this study was to use enterocin CCM 4231 to control Listeria monocytogenes contamination in ´ salami. dry-fermented Hornad

2. Materials and methods

2.1. Bacterial strains and media The strain Listeria monocytogenes Ohio (INRA, Jouy-en-Josas, France) was used for direct inoculation in a salami manufacture experiment as well as an indicator organism in the bacteriocin activity testing. This strain was cultivated in tryptone soy broth supplemented with 0.6% of yeast extract (Becton & Dickinson, Cockeysville, USA) at 30– 328C. The total and the survived numbers of listerial cells in dry fermented salami were enumerated as follows: collected samples (10 g) were vortexed in 90 ml of Ringer solution (Difco, Surrey, UK) for 5 min to be homogenized. Then, serial dilutions were prepared. And, 1 ml from the appropriate dilutions was inoculated into the tubes (every dilution in three tubes) containing Fraser broth base supplemented with Fraser broth additive (Becton & Dickinson), cultivated at 29–308C for 2 days. Then 10 ml of culture from every tube was spread plating on Oxford agar with Listeria selective supplement (Becton & Dickinson) and enumerated by the most

probable numbers (MPN) technique. To verify enumerated L. monocytogenes cells, BBL crystal Grampositive identification system (Becton & Dickinson) was used.

2.2. Bacteriocin preparation and titre determination The bacteriocin producing strain Enterococcus faecium CCM 4231 (Laukova´ et al., 1993) was grown for 18 h at 378C in Todd–Hewitt broth ˇ ˇ ´ Michal’any, Slovakia). Cells were (Imuna, Sarisske removed by centrifugation at 10 000 g for 30 min. The supernatant fluid (SF, adjusted with 5 mM EDTA and heated at 808C for 30 min) was then purified as described previously by Laukova´ et al. (1999). Briefly, SF adjusted with the addition of phosphate buffer (PB, pH 7.2) to 50 mM was applied to a phenyl-Sepharose high-performance liquid chromatography column (HPLC) equilibrated with 50 mM buffer A containing sodium-kalium phosphate (pH 7.0–7.8; saturated with 10% of solid ammonium sulphate) at a flow-rate of 30 ml h 21 . The fraction was eluted in distilled water at a flowrate of 60 ml h 21 . Tris–HCl (1 M, pH 8.0) was added to this fraction to a concentration of 20 mM. The fraction was then applied to a DEAE-Sepharose HPLC column equilibrated with buffer B containing 20 mM Tris–HCl (pH 8.0), at a flow-rate of 60 ml h 21 . The bacteriocin was eluted with buffer B adjusted by the addition of 350 mM NaCl. The bacteriocin activity was determined by the agar spot test using the critical dilution method (Mayr-Harting et al., 1972). Activity was defined as the reciprocal of the highest two-fold dilution demonstrating complete inhibitory activity of the indicator and was expressed in activity units (AU) per ml of culture medium. The bacteriocin which was immediately used for the experiment was kept at 48C. The remainder was stored at 2 208C.

2.3. Experimental salami manufacture The salami mixture contained (in kg per 1.000 kg of ready product): beef back meat without bones, 310; pork meat specially amended according to technological parameters, 515; boiled pork meat, 465; bacon, 250; nitrite curing salt, 40; black pepper, 4.40; red pepper, 0.60; cayenne pepper, 0.60; glu-

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cose, 6.10; garlic concentrate, 0.80; clove nail grind, 0.30; dextrose, 3. Starter culture (mixture of Lactobacillus and Pediococcus) was added at 25 g per 100 kg (5.0 3 10 11 cfu ml 21 ). The initial pH of the meat mixture was 6.0. The bulk salami mixture was prepared in the pilot plant and 1.5 kg for each of three trials was transferred to the laboratory for the experiments. Three independent trials were conducted, each comprising five samples. Trial A (control), comprised only the untreated salami mixture. For trial B, the salami mixture was inoculated with an 1% inoculum of Listeria monocytogenes culture (10 8 cfu ml 21 ), while for trial C 12 800 AU g 21 of enterocin CCM 4231 were added to the salami mixture inoculated with L. monocytogenes. Samples prepared this way were transferred back to the pilot plant where they were kept separately in the drying rooms for 3 weeks under different temperatures. For 1 day, the temperature in the box was 248C; the second day it was 238C; the third and fourth days it was 228C, the fifth day 218C, the sixth day 208C and the seventh day 188C. Subsequently, the temperature used was 158C. The salamis smoking procedure was performed according to technological parameters. Starting on the third day after the salamis were placed in the drying rooms the smoke shocks were used for 2.5 h daily for a period of 5 days.

2.4. Salami sampling For the microbiological determinations, the salamis were sampled with a sterile lancet, removing 10 g from the centre. The samples were then treated as described in Section 2.1. Sampling was carried out on day 1, before and after bacteriocin addition, on day 2 and after 1, 2 and 3 weeks. All samples were examined in duplicate. The bacteriocin activity in the salami samples was checked. Two-fold dilutions of homogenized samples were prepared in PB (pH 7.) The pH values of the samples were measured with a pH meter Model 3310 (Jenway, UK) equipped with a combined pH electrode.

3. Results and discussion The initial number of L. monocytogenes in the inoculated salami mixture was 10 4 cfu g 21 . Immedi-

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ately after enterocin addition, the Listeria count was reduced by log 1.67 (Fig. 1). Although on the second day, the growth of Listeriae in the presence of enterocin CCM 4231 reached 3.38 cfu g 21 (log 10), a difference 1.72 log was found compared with the control and the noticeable difference was maintained during the whole experiment (Fig. 1). After 1 week of ripening, the number of L. monocytogenes cells was 10 7 cfu g 21 in the control and 10 4 cfu g 21 when enterocin was present (Fig. 1). During the second and third week of ripening high levels of L. monocytogenes were observed but the effect of enterocin was still significant. The reference salami mixture did not contain L. monocytogenes (results not shown). The pH value at the end of experiment was 5.3 in all samples. A variety of factors can prevent the growth of microorganisms, one of which is competition by LAB present in the products (Stecchini et al., 1992). Scanell et al. (1997) reported a synergistic effect of nisin and organic acids for reduction of Salmonella kentucky and Staphylococcus aureus of fresh pork sausage. Berry et al. (1991) reported the use of a bacteriocin-producing Pediococcus spp. to control postprocessing L. monocytogenes contamination of frankfurters. Bacteriocidal effect on L. monocytogenes cells in Italian sausages was reported by Villani et al. (1997) using bacteriocin substance produced by Staphylococcus xylosus. Moreover, the inhibitory effect of enterocin CCM 4231 was reported for dairy products (Laukova´ et al., 1999). In addition, antilisterial effect of enterocin CCM 4231 was reported for other environments such as rumen fluid and cattle slurry (Laukova´ ´ 1998; Laukova´ et al., 1998). In spite and Czikkova, of the fact that the counts of L. monocytogenes were influenced by enterocin added, bacteriocin activity was not detected in the experimental salamis. One explanation for this fact could be e.g. limited diffusion of the bacteriocin into the meat mixture as was also referred to by Hugas et al. (1995). There might also be some loss of activity after a certain time. Finally, because the difficulties associated with the bacteriocin activity detection in meat products, the use of an insufficiently sensitive detection method in salami samples could be also considered. On the basis of our preliminary results it could be concluded that enterocin CCM 4231 showed a bacteriostatic effect. To achieve a more effective result as well as the complete elimination of contaminant strains, a

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´ salami. `, Experimental sample before enterocin addition; Fig. 1. Effect of enterocin CCM4231 against Listeria monocytogenes in Hornad l, experimental sample with enterocin; j, control sample.

higher concentration of purified bacteriocin may be needed. A synergistic use of two bacteriocins or use of bacteriocin together with another antagonistic system again food contaminants as reported by ´ Rodrıguez et al. (1997) should also be considered. To evaluate bacteriocin activity in meat samples, their pre-treatment according to Coffey et al. (1998) is the preferred method.

Acknowledgements The authors acknowledge that some of the results presented in this paper have been previously published (Laukova´ et al., 1999). This work was partially supported by the EU-Copernicus Project ERBCIPACT 940160.

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