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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Aims: To investigate factors influencing bacteriocin production and bacteriocin stability of the bioprotective culture Carnobacterium piscicola strain A9b.

Methods and Results: Maximum activity was obtained in MRS7 broth (MRS adjusted to pH 7·2), with or without glucose. No bacteriocin was produced in APT broth when a low inoculum level (0·001%) was used. In contrast, inoculum level did not influence bacteriocin production in BHI and MRS7 without glucose. Bacteriocin production in APT was induced by the presence of an extracellular compound present in the sterile, filtered, cell-free supernatant fluid of a stationary-phase culture. Increasing concentrations of NaCl (2–7%) reduced bacteriocin production and maximum cell density of C. piscicola A9b when grown in cooked fish juice at 4°C.

Conclusions: Media composition, inoculum level and sodium chloride concentration affected production.

Significance and Impact of the Study: The influence of NaCl on bacteriocin production may negate the inhibitory effect of C. piscicola A9b against Listeria monocytogenes in salty foods.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Listeria monocytogenes is isolated frequently from freshly-produced, cold-smoked salmon (Eklund et al. 1995; Rørvik et al. 1995; Jørgensen and Huss 1998). Due to the psychotrophic and halotolerant nature of L. monocytogenes, there are concerns about the risk of consuming contaminated cold-smoked salmon. Inoculation trials show that this pathogen grows to considerable cell numbers during cold storage (Rørvik et al. 1991; Guyer and Jemmi 1991; Rørvik and Yndestad 1991; Hudson and Mott 1993; Nilsson et al. 1997). Although cold-smoked salmon has never been implicated in human listeriosis, cases are reported from the consumption of other lightly preserved, ready-to-eat fish products such as smoked mussels, cold-smoked rainbow trout and gravad trout (Ericsson et al. 1997; Brett et al. 1998; Miettinen et al. 1999).

As L. monocytogenes is a common micro-organism in the environment, and since the cold-smoking process does not contain a listericidal step, it is impossible for all commercial processors to produce Listeria-free, cold-smoked salmon. Government and industry have focused on introducing critical control points that can prevent the growth of L. monocytogenes in ready-to-eat fish products such as cold-smoked salmon. This hazard may be controlled by using purified bacteriocins or live protective bacterial cultures (Nilsson et al. 1997, 1999; Duffes et al. 1999a, b). However, the listericidal or listeristatic efficiency of bacteriocins and protective cultures in cold-smoked salmon may be influenced by physico-chemical and microbiological factors such as NaCl (typically 3–6% in the water phase), temperature and cell density of the protective culture. The effect of NaCl on bacteriocin production and activity varies, and may be enhanced (Thomas and Wimpenny 1996; Parente et al. 1998; Uguen et al. 1999) or reduced (De Vuyst et al. 1996; Casla et al. 1996; Bouttefroy et al. 2000).

Previously, the selection and application of Carnobacterium piscicola strains as protective cultures for the control of L. monocytogenes in cold-smoked salmon was described (Nilsson et al. 1999). Strain A9b produces an antilisterial bacteriocin against L. monocytogenes in cold-smoked salmon juice. However, C. piscicola A9b only suppressed the growth of L. monocytogenes on cold-smoked salmon, possibly due to a lack of bacteriocin production or the compound binding to the food matrix. It was suggested that the inability of A9b to produce bacteriocin on cold-smoked salmon could be due to a higher NaCl content in the product, 5% water-phase salt (wps) compared with 4% wps in cold-smoked salmon juice. In the present study, bacteriocin production by C. piscicola A9b was evaluated in different laboratory media, and the influence of NaCl and inoculum level in a salmon model system was determined.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Bacterial strains

Carnobacterium piscicola A9b, which produces an antilisterial bacteriocin (Nilsson et al. 1999), was obtained from the – 80°C stock culture collection and maintained on BHI (brain heart infusion) agar (Oxoid) containing 1·2% agar (Bie & Berntsen A/S) at 5°C. A new master plate was prepared from the stored culture plate after a maximum of 6 weeks of storage. Two strains of L. monocytogenes (O57 and O49, isolated from sugar-salted salmon at the Danish Institute for Fisheries Research, Lyngby, Denmark) were used as target bacteria in the bacteriocin assays. These strains were treated and stored as strain A9b. Carnobacterium piscicola DSM20730, obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), is very sensitive to the bacteriocin produced by strain A9b, and was used as target strain to verify the absence of bacteriocin production in fish juice with NaCl.

Laboratory media and modifications

The growth of, and bacteriocin production by, C. piscicola A9b was assessed using four commercial media: All Purpose Tween (APT, Difco), Trypticase Soy Broth (TSB, Oxoid), BHI broth and deMan, Rogosa and Sharpe (MRS, Merck) broth. All media were prepared according to the manufacturer’s instructions. Growth and bacteriocin production were determined in various modifications of MRS. As Carnobacterium does not initiate growth at the low pH of MRS (pH 5·7 ± 0·2), this medium was adjusted to pH 7·2 ± 0·2 using 2 N NaOH before autoclaving (MRS7m). Despite strict adherence to time and temperature guidelines for sterilizing MRS7m, the medium often appeared dark brown and the pH had decreased from 7·2 to 6·7 after autoclaving, indicating the presence of Maillard products. A new MRS broth (MRS7) was prepared by assembling the individual components, based on the manufacturer’s formula, as autoclaved concentrated solutions, with the exception that sodium acetate trihydrate was used in place of the anhydrous sodium acetate. Growth and bacteriocin activity were determined in this medium without glucose (MRS7-G) or without Tween 80 (MRS7-T). No pH adjustment was necessary since the pH of basal media and additives was 7·0–7·2 before and after autoclaving.

Fish juice preparation

Cooked fish juice was prepared from fillets of eviscerated, frozen sea trout (Salmo trutta) with modifications to the procedure of Dalgaard (1995). The fish were thawed, filleted, chopped into chunks and weighed. Tap water was added to fish in a ratio of 1 : 4 (water : fish w/w) and boiled for 10 min. The liquid yield was 33% when separated from the solids through a kitchen strainer. The liquid was brought to a gentle boil for 5 min, remaining unstirred. Particulate material was removed using coffee filters and the filtrate was evenly distributed into culture bottles. Salt was added at final concentrations of 0–7% and the capped bottles were steamed at 100°C for 30 min. The bottles were stored at 4°C before use the next day.

Measurements during experiments

During culture incubation, growth was followed using optical density (O.D.) measurement at 600 nm (Pharmacia Biotech, Hørsholm, Denmark; Novaspec. II), and aliquots were diluted using 0·1% peptone water when the O.D. was > 0·4. The pH was recorded for culture aliquots. Additional aliquots were removed for assaying bacteriocin activity. These samples were centrifuged (10 000 g, 10 min, 25°C), adjusted to pH 6·5 ± 0·25 using pH test paper (Universalindikator, pH 0–14, Merck), sterile-filtered (0·2 μm) and stored at – 20°C in sterile Eppendorf tubes.

Bacteriocin assay

Assays were conducted using either of two L.monocytogenes strains in an agar well diffusion assay as described previously (Nilsson et al. 1997). In short, BHI plates seeded with approximately 106L. monocytogenes ml−1 were allowed to solidify and 6 mm wells punched. Aliquots of sterile-filtered supernatant fluids were added to the wells; the plates were incubated at 5°C for 18–20 h and thereafter, at 25°C for 18–22 h. Strain O57 was used in the first assays for the laboratory media experiments and strain O49 was used in all other experiments. When no bacteriocin was detected in the fish juice + NaCl trials, a more sensitive target strain (C. piscicola DSM20730) was used for verification. The pre-culture of L. monocytogenes (25°C for 24 h) was stored at 4°C until the assay was performed (up to 8 h later). Bacteriocin production was expressed as arbitrary units (AU) ml−1 and was calculated from the dilution factor and the 50 μl sample added to the wells. Specific bacteriocin activity was calculated as AU O.D.−1. Maximum bacteriocin production and maximum specific activity were determined for each growth curve.

Influence of inoculum level on bacteriocin production

Carnobacterium piscicola pre-cultured for 20–24 h in APT, BHI or MRS7-G at 25°C was added to 100 ml of each medium (APT, BHI, MRS7-G) at 1% or 0·001% final concentration. Aliquots at 18 and 24 h were removed for O.D., pH and bacteriocin assays against L.monocytogenes O49. The effect of inoculum size was determined in a cell extinction experiment in which a colony was picked to MRS7-G and incubated at 25°C for 24 h. Cells were washed twice in 0·1% peptone–0·85% NaCl and diluted 10−2–10−9 in 20 ml MRS7-G. Aliquots were removed for O.D., pH and bacteriocin assays until the O.D. values were equivalent.

Effect of culture supernatant fluid on bacteriocin production

A colony was inoculated into 10 ml APT and incubated at 25°C for 24 h. A 1% inoculum was transferred into 10 ml APT and incubated for 18 h at 25°C. This second culture was added at 1 or 0·001% inoculum levels to 50 ml fresh broth. Cells from the second culture were harvested, washed twice in 0·1% peptone–0·85% NaCl and inoculated at 1% inoculum into fresh broth. Cell-free, filter-sterilized supernatant fluid, prepared from the second culture, was added at 1% to fresh broth, followed by the 0·001% inoculum. Aliquots were removed for O.D., pH and bacteriocin assays against L. monocytogenes O49 until the O.D. values were equivalent.

Effect of NaCl on bacteriocin production in fish juice

Carnobacterium piscicola cells, pre-cultured in BHI containing 4% NaCl at 4°C for 5 days, were inoculated (1% level) in fish juice containing 0–7% NaCl. Samples were incubated at 4°C, and aliquots were withdrawn at various times during an 8 week experiment for determination of growth and bacteriocin activity against L.monocytogenes O49.

Effect of NaCl on bacteriocin stability and activity

Sterile-filtered supernatant fluid of C. piscicola grown in MRS7-G was used to determine whether salt affected pre-formed bacteriocin stability during storage. Aliquots (100 μl each) were added to sterile Eppendorf tubes containing final concentrations of 0–10% NaCl, with 10 mmol l−1 dipotassium phosphate (pH 6·2) added to a final volume of 200 μl. Tubes were stored for 1 month at 4°C and bacteriocin activity assayed against L. monocytogenes O57.

In a separate experiment, the bacteriocin assay was modified to contain final salt concentrations of 1–8% in BHI soft (0·8%) agar. The BHI soft agar control contained 0·5% NaCl. Supernatant fluids from cells grown in fish juice (containing 0–4% NaCl) were used to assay the effects of salt included in the agar diffusion assay against L. monocytogenes O49. Agars containing > 4% salt required additional incubation time to visualize the hydrolytic zones around the sample wells.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Influence of laboratory media on bacteriocin production

Carnobacterium piscicola grew and produced bacteriocin in all media with the exception of APT (Table 1). Growth in APT resulted in the highest cell density, yet no bacteriocin was produced when a 0·001% inoculum was used. The optimal media for production of bacteriocin were MRS7m, MRS7 and MRS7 omitting glucose (MRS7-G), even though the latter broth resulted in the lowest maximum cell density. Also, it was observed that bacteriocin production decreased several fold in the absence of Tween 80 in MRS7.

Table 1.   Bacteriocin production by Carnobacterium piscicola A9b (0·001% inoculum precultured in APT) in different laboratory media at 25°C. Listeria monocytogenes strain O57 was used as target organism in the bacteriocin assay Thumbnail image of

It should be noted that the dilution scheme, sequential halving to extinction, used for quantifying bacteriocin production in the well diffusion assay was dependent on the visual sensitivity of the agar-based assay. Therefore, a factor of 2, i.e. 320 AU ml−1 vs. 640 AU ml−1, between values for bacteriocin production was not considered significant.

Growth of C. piscicola A9b in MRS7-G broth at 25°C resulted in measurable production of bacteriocin from approximately O.D.600nm 0·08 (Fig. 1). Specific activity paralleled the exponential growth rate, then tapered and levelled as a second growth phase began and levelled. During rapid exponential growth, the pH decreased by 0·3 unit whereas the pH increased by 0·2 units during the second phase of growth. This could indicate de-amination of peptides and amino acids originating from 1% peptone, 0·8% meat extract and/or 0·4% yeast extract in the medium.

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Figure 1.  Growth (□), bacteriocin production (▵) and pH development (○) of Carnobacterium piscicola A9b in MRS7-G at 25°C. Growth and bacteriocin production were measured as optical density at 600 nm and specific activity (AU O.D.–1600), respectively. Strain O49 was used as target organism in the bacteriocin assays

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Effect of pre-inoculum media and inoculum level on bacteriocin production

The effect of the pre-inoculum medium and inoculum size on bacteriocin production in three different media was determined (Table 2). The production of bacteriocin in BHI and MRS7-G was unaffected by the inoculum size. In contrast, no bacteriocin production was observed in APT when a 0·001% inoculum was used, whereas a 1% inoculum resulted in bacteriocin production of 160 AU ml−1 (Table 2).

Table 2.   Influence of preinoculum medium and inoculum level on growth and bacteriocin production of Carnobacterium piscicola A9b at 25°C. Listeria monocytogenes strain O49 was used as target organism in the bacteriocin assay Thumbnail image of

An inoculum extinction experiment was performed to determine whether a lower inoculum than 0·001% influenced bacteriocin production in MRS7-G. The maximum O.D. was 0·5–0·6 for all cultures, and independent of inoculum level. Stationary phase was reached at 19–22 h for high inocula (10−2–10−4 dilutions) and at 40–46 h for more dilute inocula (10−5–10−9). Bacteriocin production was 320 AU ml−1 for the highest inocula (10−2–10−3), while production for the other dilutions was 160 AU ml−1. For MRS7-G, the inoculum level had no effect on maximum growth and maximum bacteriocin production.

Effect of culture supernatant fluid on bacteriocin production

In APT broth, bacteriocin production was not detected when a low inoculum was used, whereas a high inoculum caused bacteriocin production (Tables 1 and 2). An evaluation was made as to whether inducing factors were in the culture supernatant fluid by determining whether cell-free, filter-sterilized supernatant fluid at a level of 1% enabled the low-level inoculum (0·001%) of C. piscicola A9b to produce bacteriocin (Table 3). The production level was equivalent to that obtained when a 1% inoculum was used. The low amount of bacteriocin carryover in the filter-sterilized supernatant fluid (80 AU ml−1, resulting in 0·08 AU ml−1 initially) was insufficient to result in the higher production level observed.

Table 3.   Induction of bacteriocin production by Carnobacterium piscicola A9b in APT at 25°C. Listeria monocytogenes O49 was used as target organism in the bacteriocin assay Thumbnail image of

Effect of NaCl on bacteriocin production in fish juice

Maximum bacteriocin production in fish juice was unaffected by 1–3% NaCl (Fig. 2). However, bacteriocin production decreased inversely with increasing NaCl concentrations, and could not be detected in fish juice medium containing 6 and 7% NaCl. By using a highly sensitive target bacterium, C. piscicola DSM20730, bacteriocin was detected in fish juice containing 6% NaCl but not in 7% NaCl (data not shown). Bacteriocin production in fish juice followed maximum growth values both at 4°C (Fig. 2) and at 25°C (data not shown). Maximum growth was dependent on the salt concentration and declined inversely with increasing concentration.

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Figure 2.  Growth (a) and bacteriocin production (b) in fish juice containing 0% (□), 1% (▵), 2% (○), 3% (▴), 4% (▮), 5% (●), 6% (▿) and 7% NaCl (▾) when incubated at 4°C. Strain O49 was used as target organism in the bacteriocin assays

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Influence of NaCl on bacteriocin stability and activity

Bacteriocin stability was not affected by salt. When supernatant fluid was incubated for 1 month at 4°C in 0–10% NaCl, the activities were equivalent when assayed against L.monocytogenes O57 (data not shown).

The agar well diffusion assay was modified to investigate the effects of higher salt concentrations (Fig. 3) analogous to the concentrations found in salted and smoked salmon. A reduction in bacteriocin activity was observed in media containing between 2 and 4% NaCl. The bactericidal effect produced by C. piscicola A9b was high at 0·5 and 1% NaCl, and enhanced at 6–8% NaCl.

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Figure 3.  Bacteriocin activity of extracts from fish juice containing 0% (□), 1% (▵), 2% (○), 3% (▮) and 4% NaCl (▴). The assays against Listeria monocytogenes O49 were performed in agar containing increasing concentrations of NaCl

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Carnobacterium piscicola strain A9b has previously been shown to produce an antilisterial bacteriocin (Nilsson 2000), and it is shown in the present study that the optimal media for production of bacteriocin by C. piscicola A9b are MRS7m, MRS7 and MRS7 without glucose. Stoffels et al. (1992) found that MRS is a good medium for production of bacteriocin by C. piscicola U149. However, our MRS7-G broth avoids the Maillard reaction, which may cause initial growth inhibition. Similarly, other studies of growth and bacteriocin production in Carnobacterium spp. have shown the importance of pH modification and/or control in MRS broth (Mathieu et al. 1993; Schillinger et al. 1993; Holck et al. 1996). In other studies, growth and bacteriocin production of C. piscicola were conducted in MRS broth without 0·5% acetate, since growth of these bacteria is inhibited in the low pH broth (Schillinger and Holzapfel 1990; Schillinger et al. 1993; Khouiti and Simon 1997). However, investigations performed in this laboratory showed that acetate only influenced bacteriocin production of C. piscicola and not growth in pH-modified MRS broth (unpublished data). It was shown that bacteriocin production was reduced in MRS7 omitting Tween 80. A similar requirement for Tween 80 was found in the production of lactocin 705 (Vignolo et al. 1995).

Bacteriocin production was initiated during the exponential phase of growth in MRS7-G at 25°C, and maximum antilisterial activity was produced during the late exponential phase. Carnobacterium piscicola produces bacteriocin in the early exponential growth phase (Ahn and Stiles 1990; Stoffels et al. 1992) or mid-exponential growth phase, depending on the medium composition (Stoffels et al. 1992). The present study was in agreement with studies involving fish model systems in that glucose was not required for bacteriocin production and that the production could be sustained in media containing no carbohydrate source.

Bacteriocin production by C. piscicola A9b was independent of the inoculum level added to fresh media, except in the case of APT. The latter phenomenon could be explained by the inducing effect of an extracellular compound present in the sterile-filtered, cell-free supernatant fluid of spent APT (Table 3). Although the extracellular compound was not identified, it is surmised that bacteriocin production by C. piscicola A9b could be induced by its own bacteriocin. Autoregulation has been demonstrated in several bacteriocin-producing stains such as C. piscicola LV17 (Saucier et al. 1995; Quadri et al. 1997), Enterococcus faecium CTC492 (Nilsen et al. 1998), Lactobacillus plantarum C11 (Diep et al. 1996) and Lactococcus lactis (Kuipers et al. 1995).

Similar regulation of bacteriocin production by C. piscicola A9b was not found for cells grown in BHI and MRS7-G. This may be due to the regulation of bacteriocin production being complex, and may be influenced by pH, temperature and NaCl (Nilsen et al. 1998).

Such information is crucial to the practical application of bioprotective cultures, since early bacteriocin production gives the bacteria an advantage over food-borne spoilage and pathogenic bacteria. Furthermore, media developed to resemble the model systems are imperative for studying bacteriocin production by bioprotective cultures and determining the inhibitory mechanisms in foods.

Although Carnobacterium spp. are salt tolerant (Baya et al. 1991), maximum cell density decreased inversely to increasing NaCl concentrations in fish juice stored at 4°C. Bacteriocin production decreased in fish juice containing 2–5% NaCl while at higher NaCl concentrations, no bacteriocin production was observed. A similar inhibitory effect of NaCl has been reported for production of nisin (Bouttefroy et al. 2000), sakacin K (Leroy and De Vuyst 1999), enterocin A and B (Nilsen et al. 1998) and bavaricin (Larsen et al. 1993). Sodium chloride affects bacteriocin production either by growth inhibition, direct interference and binding of the inducing factor (Nilsen et al. 1998), or by a decrease in aw (Leroy and De Vuyst 1999).

It was shown that the bacteriocin produced by C. piscicola A9b had decreased activity in the bioassay when 2–4% NaCl was present. However, antilisterial activity at low salt (0·5 and 1%) was enhanced at high salt concentrations (6–8%). Similarly, 2–4% NaCl decreased the inhibitory effect of nisin on L. monocytogenes to a minimum level, but at low and high NaCl concentrations (near 6%), the antilisterial effect of nisin was highest (Bouttefroy et al. 2000).

The inhibitory effect of high NaCl concentrations can be a result of instability, but it was shown in the present study that bacteriocin activity was stable in the presence of salt concentrations up to 10%. The influence of NaCl on bacteriocin production may negate the inhibitory effect of the producing strain in salty foods. While addition of purified bacteriocin to cold-smoked salmon, for example, may be a reliable preservation technique, it may not be possible to guarantee bacteriocin production by bioprotective cultures in salted products. In a previous study (Nilsson et al. 1999) it was shown that L. monocytogenes was inhibited in cold-smoked salmon with 5% NaCl (water-phase salt) even though no bacteriocin was detected. Thus, inhibition of L. monocytogenes was probably due to another mechanism (Nilsson et al. 1999). Further investigations will focus on such mechanisms and the regulation of bacteriocin production. The goal is to develop preservation techniques that will be useful for enhancing the safety of lightly-preserved seafood products.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

B.H. was supported by a sabbatical from the University of Alaska Fairbanks. Financial support was obtained from the Danish Food Technology Programme.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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