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Keywords:

  • bio-control;
  • dry-fermented sausages;
  • in situ bacteriocin production;
  • Lactobacillus curvatus;
  • Lactococcus lactis;
  • Listeria monocytogenes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

Aim:  Study of the effectiveness of in situ bacteriocin production by lactic acid bacteria (LAB) to control Listeria monocytogenes in dry-fermented sausages.

Methods and Results:  Two bacteriocin-producing strains: Lactococcus lactis subsp. lactis LMG21206 and Lactobacillus curvatus LBPE were grown in a pilot scale fermentor and lyophilized to be directly used in dry sausage fermentation. A commercial starter culture (Bel'meatTM SL-25) not inhibitory to L. monocytogenes (Bac starter) was mixed (1 : 1) with each of the two lyophilized bacteriocin-producing strains to obtain starters active against the pathogen (Bac+ starter). Anti-Listeria effectiveness of the Bac+ starters was studied in dry-fermented sausages. The meat batter was experimentally contaminated with a mixture of four different strains of L. monocytogenes (102–103 CFU g−1). The results showed that L. monocytogenes did not grow in any of the contaminated batches, but no significant decrease (P > 0·05) was observed either in the positive control (no added starter culture) or in samples fermented with the Bac starter culture during the fermentation period and up to 15 days of drying. When the Bac+ starter contained Lb. curvatus LBPE, cell counts of L. monocytogenes decreased to below the detectable limit (<10 CFU g−1) after 4 h of fermentation and no survivors could be recovered by enrichment beyond day 8 of drying. When the Bac+ starter culture containing Lc. lactis LMG21206 was used, a decrease in Listeria counts to below the detectable limit was achieved after 15 days of drying.

Conclusions:  The bacteriocin-producing strains studied may be used as adjunct cultures for sausage fermentations to control the occurrence and survival of L. monocytogenes.

Significance and Impact of the Study:  Addition of the Bac+ strains, especially the Lb. curvatus strain would provide an additional hurdle to enhance the control of L. monocytogenes in fermented meat products.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

For more than two decades, Listeria monocytogenes continues to raise food safety concerns especially in ready-to-eat products where the risk of listeriosis is higher. Apart from the frequent isolation of the pathogen from meat and meat products (Soriano et al. 2001), various reports have confirmed the implication of such products in listeriosis outbreaks (Farber and Peterkin 1991; De Valk et al. 2000).

Bacteriocins or bacteriocin-producing starters were often suggested to be part of the ‘hurdles technology’ to enhance the safety and keeping quality of meat products. Bacteriocinogenic strains belonging to virtually all genera of LAB have been described to inhibit L. monocytogenesin vitro and in meat systems (Hugas and Monfort 1997; Benkerroum et al. 2003). Nonetheless, most studies on the in situ effect of bacteriocins against L. monocytogenes in meat products were carried out on single strains of the pathogen or have used high levels of contamination (103–107 CFU g−1). Significant variations in sensitivities of Listeria strains to the same bacteriocin is well documented (De Martinis and Franco, 1998) and, yet, the inoculum size was reported to affect the behaviour of L. monocytogenes under particular conditions (Gay et al. 1996). In this regard, predictive growth models demonstrated that small inoculum sizes and stressful conditions extended the lag phase of L. monocytogenes compared to high inocula (Augustin et al. 2000). Therefore, results concerning the behaviour of L. monocytogenes in meat systems obtained with high initial inoculum sizes, or through the use of a single strain of Listeria for challenge experiments may, in reality, not be valid as such foods are usually contaminated with lower number of cells (Soriano et al. 2001), and with strains which differ in their sensitivities to bacteriocins.

The present study aimed to investigate the effect of two Bac+ starters consisting of blends (1 : 1) of a commercial starter and each of two anti-Listeria bacteriocin-producing strains (Lactococcus lactis subsp. lactis LMG21206 or Lactobacillus curvatus LBPE) on L. monocytogenes in dry-fermented sausages.

Bacterial cultures and media

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

Four different strains L. monocytogenes were used for challenge experiments in dry-fermented sausages. These include L. monocytogenes ATCC7644, a human clinical isolate, obtained from the American Type Collection Culture (ATCC), and three others obtained from the Belgian Coordinated Collection of Micro-organisms (BCCM, Gent, Belgium) (strain LMG16783 serovar 4a from ovine brain, strain LMG13304 serovar 1a from mesenteric lymph nodes of guinea pig and strain LMG15139 of unknown serovar and source).

Anti-Listeria bacteriocin-producing Lc. lactis subsp. lactis LMG21206 isolated from fermented milk (Benkerroum et al. 2000, 2002) or Lb. curvatus LBPE isolated from meat (this study) were used to obtain bacteriocin-producing starters (Bac+) for sausage fermentation.

Staphylococcal strains (Staphylococcus xylosus SL-25 and S. carnosus LS-25) constitutive of commercial starter (Bel'meatTM SL-25; Beldem, Groot Bijgaarden, Belgium) and L. monocytogenes strains were stored at −20°C in trypticase soya broth (TSB, BK046HA; Biokar diagnostics, Beauvais, France) containing 25% v/v glycerol. Lactic acid bacteria (LAB) cultures including Lb. curvatus LS-25 of the commercial starter were stored in de Man, Rogosa and Sharp (MRS) broth (CM129; Oxoid Ltd, Basingstoke, Hampshire, UK) in the same conditions. Before experimental use, cells were propagated in appropriate media (1% inoculum) by overnight incubation at 37°C.

Lyophilized preparations of the bacteriocin-producing strains

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

Active lyophilized cultures of Lc. lactis LMG21206 and Lb. curvatus LBPE to be directly incorporated in the batter mixture for sausage making were obtained by growing each of the bacteriocin-producing strains in 15 l of MRS broth in a 20-l fermentor (Biolafitte & Moritz, Pierre Guerin Technologies, Lyon, France) for 12–16 h at 37°C. The fermentation was performed under controlled pH (i.e. 6·0) and with moderate agitation (100 rev min−1). The culture was then centrifuged at 5000 g for 90 min at 4°C in a Beckman Avanti J25 I centrifuge (Beckman Instruments, Fullerton, CA, USA) and the supernatant decanted. The pellets were frozen at −80°C and, then, lyophilized in a SecfroidTM lyophilizer (RK8000 96 B-D; Laboratoires Lyocentre, Aurillac, France) under negative pressure (0·2 atm) and at ambient temperature. Lyophilized cultures were ground and conditioned in packs of 20 g each. Before use, lyophilized starters were analysed for total viable cell counts (TVC) and for bacteriocin production. The TVC was performed by the standard dilution method on MRS agar (CM361; Oxoid) after incubation at 37°C for 48 h. Powders with a TVC of more than 1010 CFU g−1 and inhibitory to all strains of L. monocytogenes by in vitro tests were used in the fermentation trials. A lyophilized commercial starter (Bel'meatTM SL-25; Beldem) consisting of a combination of Lb. curvatis, S. xylosus and S. carnosus was used in bacteriocin-negative samples.

To test for bacteriocin production, lyophilized starters were activated by inoculating MRS broth (10 ml) with ca 100 mg of the starter and incubating at 30°C for 16–18 h. The cultures were then centrifuged in a microfuge at a maximum speed (23 000 g) for 10 min and the supernatant filter sterilized through a MilliporeTM membrane (0·22 μm, GSWP025; Millipore Corporation, Bedford, MA, USA). The inhibitory action of the cell-free supernatant was tested against all strains of L. monocytogenes by the well-diffusion assay (Tagg and McGiven 1971). The Listeria strains were challenged separately and in combination at equal proportions. The starter cultures were also tested against each other and against themselves. To confirm in situ bacteriocin production, a sample of fermented sausages was aseptically removed at day 5 of drying, resuspended in sterile distilled water (1 : 1) and centrifuged at 5000 g for 10 min. The supernatant was filter sterilized and tested against L. monocytogenes ATCC 7644 by the well-diffusion assay.

Sausage manufacture

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

To prepare batter mixture, the meat lean and fat (85 : 15) was cut up with a KiliaTM (Fritz Reimers GmbH, Kiel, Germany) cutter and the following additives were added per kg of mixture: 28 g salt, 0·3 g sodium nitrates, 2 g dextrose, 4 g lactose, 2 g sucrose, 4 g ginger, 4 g black pepper, 2 g garlic powder and 5 g of lyophilized starter culture. The prepared sausage mixture was stuffed into 34 mm diameter semi-synthetic casing and clipped every 12–15 cm. Prepared sausages were allowed to ferment at 30 ± 2°C with a relative humidity (RH) of 85–90% for 48 h and, then, transferred to dry at 14–16°C with 75–80% RH.

In the fermentation trials, five different batches of 2 kg each were prepared: (i) noninoculated non contaminated (negative control); (ii) no added starter culture and contaminated with a cocktail of four strains of L. monocytogenes (positive control); (iii) added Bac commercial starter culture (5 g kg−1) and L. monocytogenes cocktail (bacteriocin negative batch), and (iv and v) added Bac+ starter culture (5 g kg−1) and L. monocytogenes cocktail (test batch). In the latter batch, the Bac+ starter consisted of a mixture (1 : 1) of the commercial Bel'meatTM SL-25 starter with Lc. lactis LMG21206 or with Lb. curvatus LBPE as the producer organisms.

To contaminate appropriate sausage trials, a four-strain composite of L. monocytogenes cultures was prepared. Listeria strains were grown individually overnight at 37°C in TSB and mixed at equal proportions (1 ml each) in a sterile test tube. The mixture was then serially diluted in a saline (0·85%) solution and 1 ml of the third dilution (i.e. 10−3) was used to yield an approximate initial inoculum of 102 CFU g−1.

Sausage sampling and analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

At selected intervals during fermentation or drying, sausage segments were sampled at random to perform microbial counts and chemical analysis. For microbial counts, 10 g of an emptied segment were aseptically resuspended in 90 ml of a saline solution (0·85% NaCl) with an Ultra-thurrax® (IKA T-25; Janke and Kunkel, Staufen, Germany) and serially diluted. L. monocytogenes was enumerated after incubation for 48–72 h at 37°C on Alzoreki-Sandine-Listeria medium (ALSM) (Al-Zoreky and Sandine 1990). When no Listeria was found in a 1-ml sample of the first dilution, the enrichment procedure was performed as described by Asperger et al. (1999). A sample of 25 g was aseptically removed and inoculated into 225 ml of the enrichment selective medium of Fraser half broth (Fraser and Sperber 1988) and incubated at 30°C for 24 h followed by streaking 100 μl on the surface of ASLM agar and further incubation for 48–72 h. Typical colonies were tested for catalase production. LAB were enumerated on MRS agar (CM361; Oxoid) after 24–48 h of incubation at 30°C.

The pH of merguez sausage was determined at selected intervals during fermentation and storage with a Jenway pH meter (3310; Jenway Ltd, Essex, UK). A sample of 10 g was mixed with 10 ml of distilled water prior to pH measurement.

The weight loss of sausages was monitored by selecting at random three segments of sausages and weighing them at regular intervals. The average weight loss expressed in percentage was determined according the following formula:

  • image

where Wi is the average weight of three sausage portions at zero time (just after filling and clipping the casings); Wt is the average weight of the three portions at a given time.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

Each trial of sausage making was repeated at least twice and each determination was carried out in duplicate. Statistical analysis (analysis of variance α = 0·05 and Student's t-test) of data was performed by computations using Statistical Analysis System (SAS, Institute Inc. Cary, NC, USA) software.

In vitro antimicrobial activity testing

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

The results summarized in Table 1 show that bacteriocins produced by Lc. lactis LMG21206 and Lb. curvatus LBPE were active against all Listeria strains tested individually or in combination, while the commercial starter or its constitutive strains did not inhibit any of the Listeria cultures tested. The inhibitory activity of Lb. curvatus LBPE against Listeria strains was significantly (P > 0·05) higher than that of Lc. lactis LMG21206. In contrast, none of the constitutive strains of the commercial starter was inhibited by the bacteriocin-producing LAB, nor did they inhibit the strains of L. monocytogenes or the LAB. It is noteworthy that the inhibitory activity of Lb. curvatus LBPE against L. monocytogenes was lost after treatment with different proteases but was maintained in the presence of catalase and after pH neutralization (data not shown), suggesting that the inhibitory substance produced by the strain is of a bacteriocinogenic nature.

Table 1.  Antimicrobial activity testing of Lactococcus lactis subsp. lactis LMG21206, Lactobacillus curvatus LBPE and the commercial starter Bel'meatTM SL-25 against different strains of Listeria monocytogenes and against each other
Indicator strainInhibition zone diameter (cm)*±s.e.
Bacteriocin-producing strainsBel'meatTM SL-25
Lc. lactis LMG21206Lb. curvatus LBPE
  1. *Mean values of two determinations each in duplicate; measurements include the well diameter (8 mm).

  2. †The four cultures of L. monocytogenes mixed at equal proportions.

  3. ‡No inhibition.

L. monocytogenes LMG1513919·0 ± 0·025·0 ± 0·0
L. monocytogenes LMG1678314 ± 0·318·5 ± 0·7
L. monocytogenes LMG1330415·5 ± 0·623·5 ± 0·7
L. monocytogenes ATCC 764412·0 ± 0·020·0 ± 0·4
Mixture of Listeria strains†12 ± 0·618·5 ± 0·4
Lc. lactis LMG21206–‡14·0 ± 0·0
Lb. curvatus LBPE
Bel'meatTM SL-25

Sausage manufacture

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

To assess the suitability of the starter cultures studied herein; the main technological parameters were monitored during processing of dry-fermented sausages. The commercial starter (Bel'meatTM SL-25) was used as a reference.

Variations in pH during fermentation and drying are summarized in Fig. 1a,b, respectively. The figure shows that the pH decreased in all samples regardless of whether or not they were inoculated with the starter culture. During drying, the pH continued to decrease in all samples to reach the lowest values at day 5 and then slightly increased to be maintained stable at ca 5·3 (Fig. 1b). No significant difference (P > 0·05) was observed in pH variations between all batches over the whole period of drying.

image

Figure 1. pH evolution during fermentation (a) and drying (b) of dry-fermented sausage: negative control (noninoculated and noncontaminated with Listeria monocytogenes) ( inline image), positive control (noninoculated and contaminated with a four-strain L. monocytogenes mix) ( inline image), Bac batch (inoculated with the commercial starter; Bel'meatTM SL-25, and contaminated with the Listeria mix) ( inline image), Bac+ batch (inoculated with a starter composed of a 1:1 mixture of Bel'meatTM SL-25 and a bacteriocin-producing Lactococcus lactis subsp. lactis LMG21206, and contaminated with the Listeria mix) ( inline image), and Bac+ (inoculated with a starter composed of a 1:1 mixture of the commercial starter and a bacteriocin-producing Lactobacillus curvatus LBPE, and contaminated with the Listeria mix) ( inline image). Error bars represent the standard error for duplicate experiments

Download figure to PowerPoint

As another important technological parameter to assess the efficiency of the drying step, the weight loss was monitored as function of time and the results showed that sausage samples lost between 49 and 52% of their initial weight at day 19 of drying with a concomitant sharp decrease in the activity of water (unpublished data).

Enumerations of LAB

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

Cell counts of LAB were monitored during sausage fermentation and drying. Numbers of LAB increased regularly in all samples (including controls) during fermentation (Fig. 2a) indicating a normal sausage-making process. However, such growth was significantly (P < 0·05) slower in noninoculated samples within the first 24 h of fermentation. The counts of LAB continued to increase within the first 5 days of drying in all batches, with the exception of the batch fermented with the Bac+ starter culture containing Lc. lactis LMG21206 (Fig. 2b).

image

Figure 2. Counts of lactic acid bacteria (CFU g−1) during fermentation (a) and drying (b) of dry-fermented sausage: negative control (noninoculated and noncontaminated with Listeria monocytogenes)( inline image), positive control (noninoculated and contaminated with a four-strain L. monocytogenes mix) ( inline image), Bac batch (inoculated with the commercial starter; Bel'meatTM SL-25, and contaminated with the Listeria mix) ( inline image), Bac+ batch (inoculated with a starter composed of a 1:1 mixture of Bel'meatTM SL-25 and a bacteriocin-producing Lactococcus lactis subsp. lactis LMG21206, and contaminated with the Listeria mix) ( inline image), and Bac+ (inoculated with a starter composed of a 1:1 mixture of the commercial starter and a bacteriocin-producing Lactobacillus curvatus LBPE, and contaminated with the Listeria mix) ( inline image). Error bars represent the standard error for duplicate experiments

Download figure to PowerPoint

Behaviour of L. monocytogenes in dry-fermented sausages.

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

The results of the survival of L. monocytogenes in controls (noninoculated) and in batches inoculated with different starter cultures during fermentation and 22 days of drying are depicted in Table 2. It could be clearly seen from these data, that Listeria did not multiply in the dry-fermented sausages even when no starter culture was used. In the positive control and in the samples inoculated with the Bac starter, L. monocytogenes was inhibited, but remained viable until day 15 of drying. Thereafter, the pathogen could be recovered in the positive control by the enrichment procedure, while no such recovery was possible in samples fermented with the nonbacteriocin-producing commercial starter.

Table 2.  Numbers of Listeria monocytogenes (CFU g−1) as function of time during fermentation and drying of dry-fermented sausages
HoursFermentation
No starter added (positive control) Bac Bac+ (Lb)* Bac+ (Lc)†
05·2 × 1024·6 × 1021·1 × 1026·2 × 102
45·5 × 1021·1 × 1021 × 1027·0 × 102
122·2 × 1021·5 × 102<10‡0·5 × 102
242·0 × 1022·5 × 102<104·9 × 102
4810 × 1029·0 × 102<106·7 × 102
  1. –, Not detected in 25 g after enrichment.

  2. *The starter contains Lactobacillus curvatus LBPE as the bacteriocin-producing strain.

  3. †The starter contains Lactococcus lactis subsp. lactis LMG21206 as the bacteriocin-producing strain.

  4. ‡Not detected in 1 ml of the first dilution (10−1) but recovered by enrichment.

  5. §Time ‘zero’ of drying corresponds to 48 h of fermentation.

DaysDrying
No starter added (positive control) Bac Bac+ (Lb) Bac+ (Lc)
10 × 1029·0 × 102<106·7 × 102
52·5 × 1021·8 × 102<104·1 × 102
80·4 × 1021·9 × 102<101·2 × 102
122·3 × 1021·1 × 1020·4 × 102
150·3 × 1020·3 × 102<10
19<10
22<10

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

The present study demonstrated that Lc. lactis LMG21206 and Lb. curvatus LBPE inhibit L. monocytogenes in dry sausage fermentation by in situ bacteriocin production. The lactococcal strain was previously identified and characterized to produce a bacteriocin different from nisin (Benkerroum et al. 2000) and would belong to the class IIa bacteriocins. Lactobacillus curvatus LBPE isolated in this work was identified by a PCR-based technique (unpublished data) and was shown to produce a bacteriocin which is not yet fully characterized to be compared with known bacteriocins produced by Lb. curvatus such as curvaticin (Garver and Muriana 1994) and curvacin (Verluyten et al. 2004). Nonetheless, a preliminary characterization showed that it is stable in a wide range of pH (2–11) at room temperature and resists autoclaving at pH 8·0 or lower (unpublished data) suggesting that it is a pediocin-like bacteriocin of the class IIa bacteriocins that includes the very similar curvacin A and sakacin A produced either by Lb. curvatus or Lb. sakei (van Belkum and Stiles 2000; Chen and Hoover 2003). Nonetheless, determination of the molecular weight and the amino acid sequence are needed to provide a basis for a reliable comparison with previously known bacteriocins and to confirm its relatedness to the class II bacteriocins characterized by the presence of a YGNGVXCXXXXCXV consensus motif in their N-terminal end (Eisjink et al. 2002).

As regards the potential application of the bacteriocin-producing strains, the challenge tests in the dry-fermented sausages show that they have a potential to be used as starter cultures adjuncts for meat fermentations to enhance the safety of such meat products. They do not raise concern from antibiotic resistance/transfer public health standpoint as they are sensitive to eight different antibiotics of the most used in therapy (data not shown), they allow good processing of dry-fermented sausages and they inhibit L. monocytogenes to different extents during fermentation and drying of the sausages.

Although the pH has decreased in all samples, such a decrease occurred significantly (P < 0·05) faster in inoculated than in noninoculated samples (Fig. 1a) suggesting that the strains used in the starter culture were plying an active role in the fermentation. Nonetheless, the overall pH decrease in all samples during the fermentation was rather slow compared with that reported by Fœgeding et al. (1992) who showed that a value of 4·9 was reached in <24 h of fermentation. The direct use of lyophilized starter cultures with subsequent extension of the lag phase (Holzapfel 2002) or the specific buffering capacity of the batter mixture may explain such delayed acidification.

The final aw and pH values as well as the concentration of salts, nitrites and spices, increased the antagonism of endogenous ecological factors to microbial growth and hence lowered the opportunity for many pathogenic micro-organisms to grow in the dry-fermented sausage. Yet, they do not provide an absolute guarantee against the occurrence of some pathogens as a result of survival because of processing or postprocessing contamination. Listeria monocytogenes was able to survive in dry-fermented sausages (Johnson et al. 1990) and such survival was more likely when the final pH was >4·9 (Fœgeding et al. 1992). Therefore, the use of bacteriocn-producing starter cultures as an additional hurdle to control L. monocytogenes and prevent postprocessing contaminations remains worthwhile especially if the goal is ‘zero tolerance’.

The low growth potential of L. monocytogenes in fermented sausages is well recognized (Farber et al. 1993; Lücke 2000). Nonetheless, because of the slow rate of inactivation of L. monocytogenes in these types of sausages, counts of the pathogen may exceed 100 viable cells per gram at consumption if the raw material is heavily contaminated (Schilinger et al. 1991). For example, when meat is contaminated with more than 103 CFU g−1 and fermentation is the only means to ensure the safety of the product, L. monocytogenes was shown to survive or grow during the manufacture of dry-fermented sausage (Johnson et al. 1990; Fœgeding et al. 1992).

In the present study, use of the bacteriocin-producing strain of Lb. curvatus LBPE as part of the starter culture provided efficient control of L. monocytogenes when the batter was initially contaminated with ca 100 CFU g−1 (Table 2). When this starter culture was used, Listeria counts dropped to below the detectable level in a 1-ml sample at 12 h of fermentation and no survivors could be recovered by the enrichment procedure at day 12 of drying. Such effect could be directly related to in situ bacteriocin production. The bacteriocin was indeed shown to be present, by the well diffusion assay, in an extract from the Bac+ samples. The extract tested yielded a definite inhibition zone (average diameter 20 mm). Conversely, no such inhibition could be observed with a similar extract from samples fermented with the Bac strain or from control samples. Moreover, Listeria counts did not vary significantly (P > 0·05) throughout the whole period of fermentation and during 12 days of drying in any of the other samples. The pathogen remained viable at least until day 15. Thereafter, it was completely removed from the Bac samples and those fermented with the Bac+ starter containing Lc. lactis LMG21206. In the positive control (noninoculated), listeriae could not be enumerated but detected throughout the whole experimentation period. Numbers of L. monocytogenes decreased relatively faster in samples fermented with the Bac+ starter containing Lc. lactis LMG21206, than in those fermented with the Bac starter suggesting that bacteriocin production by Lc. lactis LMG21206 enhances somewhat the inactivation of L. monocytogenes. However, the effect of the lactococcal strain was clearly lower than that observed with Lb. curvatus LBPE. The lactobacilli, especially Lb. curvatus and Lb. sakei, have been reported to have the greatest potential to grow and produce bacteriocins in fermented sausages (De Martinis and Franco, 1998). On the contrary, Lactococcus spp. of dairy origin were reported to be less competitive in meat products and a minimum of 106–107 CFU g−1 must be added if they are to be used in meat fermentations. However, they should not be excluded de facto as their effectiveness may be enhanced in some meat products or in the presence of synergistic strains. In a previous study (Benkerroum et al. 2003), we showed that a bacteriocin-producing lactococcal strain induced further decrease in Listeria counts of ca 1·7 log units in raw merguez sausages when compared with a control and, hence, the strain was considered to be a suitable protective culture for merguez sausages.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References

This work was funded in part by the Moroccan Ministry for Higher Education, Research and Culture (grant; PARS AGRO 025) and by the cooperation agreement between Walloon Region (Division des Relations International) of Belgium and the Moroccan ministry for cooperation (Article 3.6.4). The authors wish to thank Dr M. Dehaoui for his help in the statistical analysis of data.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Bacterial cultures and media
  6. Lyophilized preparations of the bacteriocin-producing strains
  7. Sausage manufacture
  8. Sausage sampling and analysis
  9. Statistical analysis
  10. Results
  11. In vitro antimicrobial activity testing
  12. Sausage manufacture
  13. Microbiological study
  14. Enumerations of LAB
  15. Behaviour of L. monocytogenes in dry-fermented sausages.
  16. Discussion
  17. Acknowledgements
  18. References
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