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Keywords:

  • cell-adsorbed bacteriocin;
  • Escherichia coli;
  • essential oil;
  • Lactobacillus curvatus;
  • Listeria monocytogenes;
  • pork meat;
  • Salmonella Typhi

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Aims:  To study the effectiveness of a combination of cell-adsorbed bacteriocin (CAB; a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments) of a Lactobacillus curvatus strain with oregano or savory essential oil to control Listeria monocytogenes in pork meat at 4°C.

Methods and Results:  The antimicrobial activity of the CAB and six different essential oils was tested by the well diffusion assay against L. monocytogenes M, Escherichia coli 10536 and Salmonella serotype Typhi CWBI-H1. The anti-Listeria activity of the CAB and oregano or savory essential oils was also investigated in pork meat. The results of the well diffusion assay showed that CAB was only inhibitory to L. monocytogenes while savory and oregano essential oils were the most active against the three indicator bacteria. In pork meat, Listeria counts have declined from c. 102 CFU g−1 to below the detectable limit during the first week of storage in samples treated with CAB or oregano essential oil and in those treated with CAB combined with oregano or savory essential oil. However, the counts of L. monocytogenes have increased after the third week of storage in all samples with the exception of those treated with the combination of CAB and oregano essential oil. The combination of CAB with savory essential oil resulted in a 2-week delay of the growth rebound compared with samples treated with CAB alone.

Conclusions:  Addition of oregano or savory essential oil exhibited a synergistic effect with CAB to control L. monocytogenes in pork meat during storage at 4°C.

Significance and Impact of the Study:  The combination of CAB with oregano or savory essential oil may be effectively used in meat industry to enhance the safety and stability of meat products.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Listeria monocytogenes is a foodborne pathogen of concern to food safety that has been isolated from pork meat (Nesbakken et al. 1996). Furthermore, the implication of pork-meat products in a number of listeriosis outbreaks (Frye et al. 2002; Mayrhofer et al. 2004) emphasizes the need to search for alternative means to control effectively the pathogen in these products.

The potential of bacteriocins of lactic acid bacteria as biological food-grade preservatives is well established (Chen and Hoover 2003; Cotter et al. 2005). However, such an application faces some limitations with respect to their narrow spectrum of action or their possible inactivation in food systems by proteases or as a result of their interactions with food constituents (Jung et al. 1992). In addition, loss of antimicrobial activity of bacteriocins against sensitive strains during food storage by spontaneous emergence of resistant mutants has been demonstrated (Rekhif et al. 1994).

Empirical application of various aromatic plants in food preservation has long been known; however, the role of the essential oil fractions of these plants in the inhibition of spoilage and pathogenic micro-organisms has been scientifically demonstrated only by the middle of the last century (Boyle 1955). At present, the antimicrobial activity of essential oils is well established, and their potential as biological adjuncts in food preservation is attracting increased research interest.

The multiple hurdles technology has been established as an efficient means to improve the safety and keeping quality of foods (Leistner 1996). This approach is even more attractive that it advocates the combination of natural antimicrobial compounds with conventional food preservation treatments to minimize the use of chemical additives in response to the growing consumers’ demand with respect to their perception of ‘natural foods.’

The effect of various combinations of essential oils or their active constituents with other antimicrobial substances has been extensively investigated (Periago et al. 2001; Valero and Francès 2006). A synergetic action between essential oils or their constituents and bacteriocins such as nisin to inhibit spoilage and pathogenic micro-organisms has been demonstrated in vitro (Ettayebi et al. 2000; Thomas and Isaak 2006). However, few studies to our knowledge have been carried out on such a synergy in food systems.

The present study aimed to investigate the antimicrobial activity of a cell-adsorbed bacteriocin (CAB) of Lactobacillus curvatus CWBI-B28 (i.e. a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustment) and selected essential oils among those commonly used in meat industry against Listeria monocytogenes, Escherichia coli and Salmonella enterica serotype Typhi by in vitro tests. The potential of CAB alone or in combination with oregano or savory essential oils to control L. monocytogenes was also investigated in pork meat during storage at 4°C.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Micro-organisms and media

Lactobacillus curvatus CWBI-B28, a bacteriocin-producing (Bac+) strain previously isolated from meat (Benkerroum et al. 2005) and shown to produce three distinct bacteriocins (Ghalfi 2006a), L. monocytogenes M, a bacon isolate sensitive to the Bac+ strain, and E. coli ATCC 10536 and S. Typhi CWBI-H1 (Centre Wallon de Bio-Industries collection, Gembloux, Belgium) were used in this study. The Bac+ strain was grown in de Man, Rogosa and Sharp broth (MRS; Oxoid, Basingstoke, UK). Listeria monocytogenes and S. Typhi were grown in trypticase soy broth (TSB; Oxoid) and E. coli ATCC 10536 was grown in lactose broth (Oxoid). All strains were stored at −80°C in their respective media with added glycerol (40%).

Working cultures were maintained at 4°C on slants of MRS agar, PALCAM agar (Oxoid), violet red bile lactose agar (VRBL; Biokar Diagnostics, France) and trypticase soy agar, for Lact. curvatus, L. monocytogenes, E. coli and S. Typhi, respectively. Before use, the strains were propagated twice by an overnight incubation at 37°C in their respective broth media.

Essential oils

Essential oils used in this study are derived from clove, coriander, oregano, savory, thyme and rosemary. They were obtained from PranaromTM (Horrues, Belgium) and stored at room temperature, protected from light and air.

Preparation of lyophilized CAB

The CAB of Lact. curvatus CWBI-B28 was obtained as described previously (Ghalfi et al. 2006b). Briefly, Lact. curvatus CWBI-B28 was cultivated at 37°C in MRS broth (12 l) in a 15-l fermentor (Biolafite, France). The fermentor was operated without aeration, with moderate agitation (80 rev min−1), and at controlled pH and temperature. The pH was maintained constant at 6·5 by automatic addition of 6 N NaOH. At this pH value, the bacteriocin of Lact. curvatus CWBI-B28 was shown to adsorb maximally onto the producer cells (Ghalfi et al. 2006b). After 12 h of fermentation, the culture was heated at 70°C for 25 min and centrifuged at 11 000 g for 25 min in a Beckman centrifuge (Avanti J-25I; Beckman, CA, USA). The cell pellets were recovered and a part (2 l) of the supernatant was filter-sterilized through a 0·22-μm MilliporeTM membrane to obtain a cell-free supernatant (CFS). The recovered pellets were re-suspended in 1800 ml of the CFS and the suspension was held at 4°C for 30 min to allow maximum bacteriocin adsorption onto the producer cells and then centrifuged at 11 000 g for 25 min. The pellets were harvested as a CAB and lyophilized (Koeltechniek Louw B.V.B.A, Rotselaar, Belgium), then stored at −20°C as an active powder until needed.

Antibacterial activity of CAB and different essential oils against selected gram-positive and gram-negative foodborne pathogens

Well diffusion assay Antimicrobial activity of CAB and essential oils against L. monocytogenes M, E. coli ATCC 10536 and S. Thyphi CWBI-H1 was tested by the well diffusion assay (Tagg and McGiven 1971). Wells (∼6 mm in diameter) were punched in plates of M17 agar (Oxoid) seeded with 0·1 ml of an active culture of each indicator strain and filled with 60 μl of CAB suspension (0·2 g of the active powder in 1 ml of sterile distilled water), or 5 μl of each of the essential oils. The plates were then incubated at 37°C for 16–18 h and the diameters of the inhibition zones surrounding the wells were measured.

Determination of minimal bactericidal concentrations against Listeria monocytogenes The minimal bactericidal concentrations (MBC) of two essential oils (i.e. savory and oregano) that gave the largest diameter of inhibition zones against L. monocytogenes M by the well diffusion assay (Table 1) were determined in liquid media as described by Wan et al. (1998). In a series of test tubes each containing 10 ml of MRS broth, savory or oregano essential oil was added to different concentrations ranging from 0 to 5·0 μl ml−1. Tubes were then inoculated with 100 μl of an overnight culture of L. monocytogenes M and incubated aerobically at 37°C for 48 h and under agitation (120 rev min−1) to keep the medium homogeneous. A 1-ml sample was withdrawn at 0 h and at 48 h, to determine the numbers of L. monocytogenes on PALCAM agar after incubation at 37°C for 48 h. The MBC was defined as the lowest concentration required for complete inhibition of the test organism after 48 h of incubation in the presence of the essential oil.

Table 1.   Diameter of the zones of inhibition (mm ± SD)* of cell-adsorbed bacteriocin (CAB)† of Lactobacillus curvatus CWBI-B28 and selected essential oils (EO) against Listeria monocytogenes M, Salmonella serotype Typhi and Escherichia coli strains
Antimicrobial agentL. monocytogenes MS. Typhi CWBI-H1E. coli ATCC10536
  1. *Results include the diameter of the well (6 mm).

  2. †CAB, cell-adsorbed bacteriocin.

  3. ‡No inhibition.

CAB26 ± 2NI‡NI
Coriander EO8 ± 115 ± 08 ± 0
Savory EO25 ± 125 ± 023 ± 1
Clove EO11 ± 114 ± 010 ± 0
Oregano EO25 ± 023 ± 023 ± 1
Rosemary EONI9 ± 110 ± 2
Thyme EO10 ± 011 ± 213 ± 1

Antilisterial activity of CAB in pork meat with or without added oregano or savory essential oil

Challenge experiments Two independent trials were conducted to study the effectiveness of the CAB to control L. monocytogenes in pork meat in presence or absence of oregano or savory essential oil. In each trial, a meat block of 1·5 kg was sliced aseptically into portions of approximately 300 g each to make five different batches (B1 to B5). The batches were placed individually in sterile aluminum foil under a laminar flow hood (Clean Air, VWR, Belgium) for subsequent treatments. Batches B2 to B5 of each trial were artificially contaminated with L. monocytogenes to ∼102 CFU g−1. The batch B1 was not contaminated and served as a negative control. After inoculations, 3 ml of CAB suspension (1 g of active powder in 5 ml of sterile distilled water) were applied to each of the batches B3 and B5. The essential oil (oregano or savory) was added to batches B4 and B5 to a final concentration of 50 μl 100 g−1 of pork meat. For practical reasons (i.e. low volume of the essential oil to be applied), the essential oil was mixed to the required volume of the CAB suspension (B5) or to an equal volume (3 ml) of sterile distilled water (B4) before application to meat portions. The batch B2 served as a positive control and, hence, was inoculated with L. monocytogenes without further treatments. All samples were placed in separate sterile plastic bags, which were then heat-sealed and held at 4°C during the course of the experiments.The trials differed only in the essential oil used in the treatment of batches B4 and B5; while oregano essential oil was used in one of the trials, savory essential oil was used in the other.

Microbiological analyses and arbitrary unit determinations Samples (20 g) were aseptically taken at 0 h weekly from each batch for Listeria enumerations. The samples were placed in sterile plastic bags containing 180 ml of sterile saline solution (0·85% NaCl) and thoroughly homogenized by hand massaging and shaking for 1 min. Suspensions were then serially diluted and a volume of 0·1 ml from each dilution was spread-plated in duplicate onto PALCAM agar (Oxoid). The CFU of L. monocytogenes were determined after incubation at 37°C for 48–72 h. Suspensions obtained from samples treated with CAB were centrifuged and the supernatant was filter-sterilized to determine the bacteriocin activity in arbitrary units (AU) as previously described (Benkerroum et al. 2005).

Statistical analysis

Each trial was repeated twice and each determination was performed in duplicate. Statistical analysis was made by analysis of variance α = 0·05% and Student's t-test.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

Antibacterial activity of CAB and various essential oils

Table 1 summarizes the results of the antibacterial activity of the CAB and selected essential oils against L. monocytogenes M, E. coli ATCC 10536 and S. Typhi CWBI-H1. As expected, the CAB was only active against L. monocytogenes M while different performances of the essential oils were recorded depending on the bacterial strain. The antibacterial activity of coriander, clove, rosemary and thyme varied greatly depending on the indicator strain, but their antilisterial activity was weak. Oregano and savory essential oils had the highest inhibitory activity against all bacteria tested and were, therefore, retained for the next experiments. The MBC of each of the latter essential oils was determined against L. monocytogenes which was completely inactivated after 48 h of incubation in presence of savory or oregano essential oil at concentrations ranging from 0·5 to 5·0 μl ml−1. In contrast, the pathogen grew well in the positive control (i.e. no added essential oil) to reach 1·7 107 CFU ml−1 after 48 h of incubation. The level of 0·5 μl ml−1 was, therefore, considered as the MBC for both savory and oregano essential oils.

Antilisterial activity of CAB in presence or absence of essential oil in pork meat

The results of the antilisterial activity of the CAB in presence or absence of oregano or savory essential oil during cold storage of pork meat are summarized in Table 2. A significant (P< 0·05) reduction in Listeria counts was observed in the first week of storage in all test samples as compared with the positive control (i.e. no added CAB or essential oil). However, the extent of this inhibition and the subsequent protection of the meat product from the growth of L. monocytogenes varied according to the treatment applied. In batches treated with the CAB in presence or absence of oregano or savory essential oil and in those treated with only oregano essential oil, the counts of L. monocytogenes have declined to an undetectable level in a 1-g sample during the first week of storage. In samples treated with savory essential oil, a reduction of c. 2 log CFU g−1 in Listeria counts was observed at the third week of storage but the CFU did not fall below the detectable level throughout the whole period of the study (Table 2). Furthermore, in all test samples, with the exception of those treated with the combination of CAB and oregano oil, an increase in Listeria counts was noted after they have reached their lowest values. Such an increase (i.e. rebound phenomenon) was observed a week earlier in samples treated with CAB alone compared with those treated with oregano essential oil alone or with the combination of CAB and savory essential oil.

Table 2.   Enumeration of Listeria monocytogenes as function of time in pork meat treated with cell-adsorbed bacteriocin (CAB) in presence or absence of oregano essential oil (OEO) or savory essential oil (SEO), and bacteriocin production (AU g−1*) in meat samples treated with CAB
Time (weeks)log10 CFU g−1 ± SDAU g−1
ControlCABOEOSEOCAB + OEOCAB + SEO
  1. *AU (arbitrary unit) is defined the reciprocal of the highest dilution giving a definite zone of inhibition by the well diffusion assay (Tagg and McGiven 1971) on a lawn of L. monocytogenes.

02·90 ± 0·152·90 ± 02·78 ± 03·0 ± 0·302·78 ± 02·78 ± 01800 ± 0·00
14·81 ± 0·180 ± 00 ± 02·11 ± 0·100 ± 00 ± 0510 ± 55·51
24·39 ± 0·30 ± 00 ± 01·70 ± 0·30 ± 00 ± 0370 ± 64·38
32·40 ± 0·20 ± 00 ± 00·78 ± 0·20 ± 00 ± 0221 ± 8·06
41·75 ± 0·131·31 ± 0·070 ± 01·70 ± 0·20 ± 00 ± 0199 ± 2·65
51·64 ± 0·212·27 ± 0·070·85 ± 0·21·81 ± 0·10 ± 00·85 ± 0·36170 ± 13·95
61·39 ± 0·072·58 ± 0·051 ± 0·172 ± 0·160 ± 01 ± 086 ± 46·32

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

In vitro antimicrobial activity assays showed that the CAB of Lact. curvatus CWBI-B28 and six different essential oils inhibited various bacterial strains of health and spoilage significance. While the CAB was only inhibitory to L. monocytogenes, the essential oils inhibited the indicator strains to various degrees depending on the essential oil and the bacterial strain (Table 1). No evidence for the difference in sensitivity among gram-positive and gram-negative bacteria to the essential oils could be noted in agreement with earlier reports (Deans and Ritchie 1987; Dorman and Deans 2000). In contrast, other studies suggested that essential oils are generally more active against gram-positive than gram-negative bacteria (Lambert et al. 2001; Delaquis et al. 2002; Harpaz et al. 2003). The high inconsistencies in the chemical composition of an essential oil depending on physiological and ecological conditions of the producing plants (Dorman and Deans 2000) may explain the variability in the susceptibility of sensitive strains regardless of their Gram staining. Yet, essential oils containing high levels of carvacrol were reported to be the most inhibitory to microbial growth (Cosentino et al. 1999). This is consistent with the fact that oregano and savory essential oils had the highest antimicrobial activity against all bacteria tested in this study, as carvacrol represents 80% (Angelini et al. 2003) and 57% (van der Mheen 2006) of these essential oils, respectively.

Determinations of the MBC of savory and oregano essential oils showed that low levels of these essential oils effectively inactivate sensitive bacteria. In vitro bactericidal effect of essential oils at low concentrations is well documented (Cosentino et al. 1999; Gill et al. 2002; Burt 2004). However, the concentrations to be used in actual food systems for preservation were shown to be as high as 100-fold of those recorded in in vitro studies (Mendoza-Yepes et al. 1997), which may affect the sensory attributes of foods. Therefore, the search for adequate combinations of essential oils with other hurdles to microbial growth have been the focus of tremendous work in recent years aiming to improve their overall effectiveness in food preservation and, hence, reduce the amount to be added.

The study of the in situ effect of CAB alone or in combination with savory or oregano essential oil on the growth of L. monocytogenes in pork meat showed different performances with overall tendency to reduce the counts of the pathogen. However, after an initial decrease in Listeria counts to their lowest levels in all the treated samples, the growth of the pathogen was re-initiated in most of them. The occurrence of this rebound phenomenon upon extended period of storage has been reported previously (Motlagh et al. 1992; Davidson and Harrison 2002). In the case of the CAB-treated samples, the steady decrease in the bacteriocin activity during storage may explain the resuscitation of L. monocytogenes when the AU have attained too low levels to inhibit the pathogen (Table 2). Inactivation of bacteriocins in meat products has been attributed to indigenous or microbial proteases (Chumchalova et al. 1998; Davidson and Harrison 2002). Nonetheless, such a rebound may not be explained solely by the bacteriocin inactivation, as the growth re-initiation was also observed in the batches treated with only the essential oils. Adaptation of the pathogen to sublethal injury (Lou and Yousef 1997; Faleiro et al. 2003), spontaneous development of resistant mutants (Davidson and Harrison 2002), interactions with food constituents (Jung et al. 1992; Mejlholm and Dalgaard 2002) and the ability of some bacteria to repair damaged cells (Gill et al. 2002) may also account for such a phenomenon. In this study, the rebound was not observed in samples treated with the combination of CAB and oregano essential suggesting a strong synergistic action between these antimicrobial compounds. A synergy action was also noted in the samples treated with the combination of CAB and savory essential oil; however, this combination failed to prevent the growth rebound which was only delayed from the third to the fourth (samples treated with only savory essential oil) or the fifth (samples treated with only CAB) week of storage.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

The combination of a CAB with oregano or savory essential oil has improved the control of L. monocytogenes in pork meat as compared with their utilization separately. However, the synergistic action of CAB with oregano essential oil provided more efficient and a longer protection of pork meat from the resuscitation of L. monocytogenes. Therefore, the combination of CAB of Lact. curvatus and oregano essential oil may be a useful means to enhance the safety and stability of meat products.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References

This work was funded by the cooperation agreement between Walloon Region (Division des Relations International) of Belgium and the Moroccan Ministry for Cooperation (Article 3·6·4).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgements
  9. References
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