Carnobacterium Strains Research Articles

Polar-Region Soils as Novel Reservoir of Lactic Acid Bacteria from the Genus Carnobacterium.

Polar habitats offer excellent sites to isolate unique bacterial strains due to their diverse physical, geochemical, and biological factors. We hypothesize that the unique environmental conditions of polar regions select for distinct strains of lactic acid bacteria (LAB) with novel biochemical properties. In this study, we characterized ten strains of psychrotrophic LAB isolated from hitherto poorly described sources-High Arctic and maritime Antarctic soils and soil-like materials, including ornithogenic soils, cryoconites, elephant seal colonies, and postglacial moraines. We evaluated the physiological and biochemical properties of the isolates. Based on 16S rRNA and housekeeping genes, the four LAB strains were assigned to three Carnobacterium species: C. alterfunditum, C. maltaromaticum, and C. jeotgali. The remaining strains may represent three new species of the Carnobacterium genus. All isolates were neutrophilic and halophilic psychrotrophs capable of fermenting various carbohydrates, organic acids, and alcohols. The identified metabolic properties of the isolated Carnobacterium strains suggest possible syntrophic interactions with other microorganisms in polar habitats. Some showed antimicrobial activity against food pathogens such as Listeria monocytogenes and human pathogens like Staphylococcus spp. Several isolates exhibited unique metabolic traits with potential biotechnological applications that could be more effectively exploited under less stringent technological conditions compared to thermophilic LAB strains, such as lower temperatures and reduced nutrient concentrations. Analysis of extrachromosomal genetic elements revealed 13 plasmids ranging from 4.5 to 79.5 kb in five isolates, featuring unique genetic structures and high levels of previously uncharacterized genes. This work is the first comprehensive study of the biochemical properties of both known and new Carnobacterium species and enhances our understanding of bacterial communities in harsh and highly selective polar soil ecosystems.

Membrane lipid composition of Carnobacterium maltaromaticum CNCM I-3298, a highly cryoresistant lactic bacterium

The growing consumption of fermented products has led to an increasing demand for lactic acid bacteria (LAB), especially for LAB tolerant to freezing/thawing conditions. Carnobacterium maltaromaticum is a psychrotrophic and freeze-thawing resistant lactic acid bacterium. The membrane is the primary site of damage during the cryo-preservation process and requires modulation to improve cryoresistance. However, knowledge about the membrane structure of this LAB genus is limited. We presented here the first study of the membrane lipid composition of C. maltaromaticum CNCM I-3298 including the polar heads and the fatty acid compositions of each lipid family (neutral lipids, glycolipids, phospholipids). The strain CNCM I-3298 is principally composed of glycolipids (32%) and phospholipids (55%). About 95% of glycolipids are dihexaosyldiglycerides while less than 5% are monohexaosyldiglycerides. The disaccharide chain of dihexaosyldiglycerides is composed of α-Gal(1−2)-α-Glc chain, evidenced for the first time in a LAB strain other than Lactobacillus strains. Phosphatidylglycerol is the main phospholipid (94%). All polar lipids are exceptionally rich in C18:1 (from 70% to 80%). Regarding the fatty acid composition, C. maltaromaticum CNCM I-3298 is an atypical bacterium within the genus Carnobacterium due to its high C18:1 proportion but resemble the other Carnobacterium strains as they mostly do not contain cyclic fatty acids.

Isolation, Identification and Characterization of Thermoduric Psychrotrophic Bacteria from Market Samples of Pasteurized Milk Collected from Mannuthy, Kerala State, India

Aims: To isolate, identify and characterize thermoduric psychrotrophic bacteria from locally available market samples of pasteurized milk.
 Study Design: Market samples of pasteurized milk were collected, subjected to laboratory pasteurization and thermoduric psychrotrophic bacteria were enumerated after 10 days of storage at 7ºC.The isolates obtained were identified by 16S rRNA sequencing and subsequent blasting. The isolates were characterized in terms of haemolytic, lipolytic and proteolytic activities. Selected isolates were also assessed for their decimal reduction time at 63ºC. 
 Place and Duration of Study: Verghese Kurien Institute of Dairy and Food Technology, (Formerly College of Dairy Science and Technology), Mannuthy, Thrissur, Kerala between September, 2019 to March, 2021.
 Methodology: A total of 42 market samples of pasteurized milk belonging to seven different brands were assessed for their thermoduric psychrotrophic bacterial population. The thermoduric psychrotrophic bacterial isolates were identified by 16S rRNA sequencing and the sequences obtained were searched with the NCBI BLAST program (http://www.ncbi.nlm.nih.gov) for their closest relatives/reference strains with a homology of over or equal to 99 per cent. Haemolytic, proteolytic and lipolytic activities of the isolates were determined by streaking them on blood agar, skim milk agar (SMA) and tributyrin agar respectively and grading them based on the presence or absence of zone of clearance around the colonies developed. Decimal reduction time at 63ºC of two selected isolates was also determined.
 Results: Six thermoduric psychrotrophic bacteria were isolated from the 42 market samples of pasteurized milk i.e., only 14.3 % of tested samples had thermoduric psychrotrophic bacterial population. 16S rRNA sequencing and subsequent blasting identified the isolates as Aeromonas caviae, Moraxella osloensis, Delftia tsuruhatensis, Staphylococcus arlettae and two strains of Carnobacterium maltaromaticum. On assessing the haemolytic activity of the isolates, A.caviae DMV01 exhibited α haemolytic activity whereas Staphylococcus arlettae DMV02, M. osloensis DMV03, C. maltaromaticum DMV05 and C. maltaromaticum DMV06 exhibited ϒ haemolytic activities. A. caviae DMV01 was found to be lipolytic and the two Carnobacterium strains exhibited proteolytic activities. Staphylococcus arlettae DMV02 was found to be both proteolytic and lipolytic. Aeromonas caviae DMV01 exhibited D63 value of 4 minutes 38 seconds whereas for Moraxella osloensis DMV03 it was 25 minutes 18 seconds.
 Conclusion: Exhibition of heat resistance by microorganisms capable of low temperature growth presents quite a challenging situation in terms of quality assurance and shelf life extension of pasteurized milk. So it is high time that dairy industry takes up initiatives to remain updated with the changes happening in pasteurized milk microflora in order to address the challenges such microorganisms may pose in future.

Carnobacterium Species Capable of Growth at Pressures Ranging Over 5 Orders of Magnitude, from the Surface of Mars (103 Pa) to Deep Oceans (107 Pa) in the Solar System.

Several permanently cold solar system bodies are being investigated with regard to their potential habitability, including Mars and icy moons. In such locations, microbial life would have to cope with low temperatures and both high and low pressures, ranging from ∼102 to 103 Pa on the surface of Mars to upward of ∼108-109 Pa in the subsurface oceans of icy moons. The bacterial genus Carnobacterium consists of species that were previously shown to be capable of growth in the absence of oxygen at low temperatures and at either low pressure or high pressure, but to date the entire pressure range of the genus has not been explored. In the present study, we subjected 14 Carnobacterium strains representing 11 species to cultivation in a complex liquid medium under anaerobic conditions at 2°C and at a range of pressures spanning 5 orders of magnitude, from 103 to 107 Pa. Eleven of the 14 strains showed measurable growth rates at all pressures tested, representing the first demonstration of terrestrial life forms capable of growth under such a wide range of pressures. These findings expand the physical boundaries of the capabilities of life to occur in extreme extraterrestrial environments.

Comparison of the performance of the biofilm sampling methods (swab, sponge, contact agar) in the recovery of Listeria monocytogenes populations considering the seafood environment conditions

AimsThe aim of this study was to evaluate the performance of sampling methods [contact plates, sponges, and swabs] in the recovery of biofilm Listeria monocytogenes populations considering the seafood environment conditions (nature of conditioning, of materials and bacterial species). Methods and resultsDifferent materials (stainless steel, polyvinyl chloride, polyurethane) were conditioned with two fish filtrates, the ready-to-eat the most consumed in Europe (smoked salmon, cod). After, we added the suspension of Listeria monocytogenes, alone or with Pseudomonas fluorescens or Carnobacterium strains, and incubated for 48 h at 8 °C. Then, the 48 h-biofilms were sampled with different methods (contact plates, sponges, and swabs). The cultivable bacterial populations were enumerated on agar, while the L. monocytogenes total and viable populations were quantified by qPCR and propidium monoazide-qPCR (PMA-qPCR), respectively. The amount of L. monocytogenes in biofilms was affected only by the nature of the conditioning with lowest adherent bacteria with cod versus with smoked salmon conditioning. Considering the amount of total population, the swab displayed the lowest values versus the sponges and the contact plates. An explanation was that the observations of the swab by microscopy showed the bacteria trapped within it. The recovery of cultivable bacterial populations was not significantly different with the three sampling methods. On the contrary, we showed that the VBNC populations were only detached by two of three methods (contact plates, sponges) while for the dead populations, those were contact plates and swabs. ConclusionsThe nature of the conditioning influenced the amount of the bacteria in biofilms. And the performance of the recovery of the bacterial populations (dead, VBNC, cultivable) was dependent on the methods used. Significance and impact of the studyThis study showed that the seafood environmental conditions influenced the biofilm formation and the assessment of the efficiency of cleaning and disinfectant operations could be significantly affected by the used sampling methods.

Growth of Carnobacterium spp. isolated from chilled vacuum-packaged meat under relevant acidic conditions

Carnobacterium spp. are frequently isolated from vacuum-packaged (VP) meat. Specific strains of Carnobacterium and their growth characteristics may be associated with the storage life of such products. This study investigated the growth of 44 Carnobacterium isolates obtained from VP meat cuts produced at three Canadian abattoirs (A, B and C) under the following conditions: pH 5.4, 6.2 and 7.4; lactic acid at 60 and 90 mM; acetic acid at 33.6 mM. Whole genome sequencing was performed for all 44 isolates and a core genome phylogenetic tree was created to identify strain variability among isolates from different abattoirs. The isolates were clustered into 11 groups. All isolates from abattoirs B and C were identified as C. divergens, while the isolates from abattoir A included both C. maltaromaticum and C. divergens at equal proportions. C. divergens isolates from abattoir A belonged to two phylogenetic groups and none of them was found in the phylogenetic groups containing isolates from abattoirs B or C. Whole genome sequencing revealed that identical strains were isolated from different samples obtained at the same abattoir. The mean growth rate and maximum population density of the C. maltaromaticum isolates were lower than those of the C. divergens isolates. C. divergens isolates from abattoir A had higher growth rates and maximum population density than those from abattoirs B and C. In conclusion, growth characteristic and whole genome analysis both demonstrated strain variability of Carnobacterium among abattoirs, which could be a result of the difference in the antimicrobial interventions used for carcasses at different abattoirs, and may be associated with different storage lives of VP meats produced from different abattoirs.

Draft Genome Sequences of Nine New Carnobacterium maltaromaticum Strains Isolated from Diseased Sharks.

ABSTRACTHere, we report the draft genome sequences of 9 strains of Carnobacterium maltaromaticum (SK_LD1 to SK_LD3 and SK_AV1 to SK_AV6), a member of the Carnobacteriaceae family (phylum Firmicutes). These strains were isolated from the brain and the inner ear of three diseased thresher sharks and two diseased salmon sharks. The genome assembly resulted in an average of 3,306,205.9 ± 29,143.9 bp and 3,085 ± 32.67 coding DNA sequences (CDS).

Effect of abattoir and cut on variations in microbial communities of vacuum-packaged beef

This report builds on the earlier studies of the shelf-life of chilled Australian vacuum packaged (VP) beef primals (striploin and cube roll), products distinguished in the global marketplace for unusually long shelf-life. Notable findings in those studies were a shelf-life of at least 26weeks at −0.5°C, low microbial counts, and relatively high sensory scores. However, growth rates for total viable counts (TVC) and lactic acid bacteria (LAB) varied among the different abattoirs. The present study adds to these findings, by providing greater definition about temporal changes in bacterial communities using terminal restriction fragment length polymorphism (TRFLP) and clone library analyses of 16S ribosomal RNA (16S rRNA) gene, and measuring statistical associations among abattoir, beef cut, storage time and sensory attributes. Bacterial communities changed over time, with Carnobacterium spp. typically predominating (29–97%) at the end of storage. Variation in TRFLP profiles showed that different Carnobacterium strains predominated in different abattoirs, and that additional variation was due to the presence of other taxa typical of VP meat microbiomes. TRFLP-based community structure correlated significantly (P≤0.01) with sensorial characteristics, such as vacuum integrity, confinement odour, and intact pack appearance of beef. This study shows that Carnobacterium spp. predominate on extended shelf-life VP beef primals, while other taxa may produce subtle effects on shelf-life duration.

English

Thirty-seven (37) enterococcal isolates were recovered from the skin and intestines of the sea bream (Sparus aurata), the most economically important fish species of the Mediterranean sea from Tunisian fish farming sites, to investigate their antimicrobial potential. All isolates were identified to the species level using genotypic tools. An investigation employing 16S rDNA sequencing in combination with randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) highlighted the predominance of the Enterococcus faecium (46%) and E. faecalis (19%) species. Other species, such as E. sanguinicola (3 strains), E. casseliflavus (3 strains), E. gallinarum (2 strains), Carnobacterium sp. (1 strain), Aerococcus viridans (2 strains) and Vagococcus carniphilus (2 strains) were also identified. The susceptibility to different antibiotics in addition to the antibacterial activities were investigated for all species identified. The isolates were sensitive to vancomycin but were resistant to several antibiotics relevant for therapy in human and animal medicine. Antibacterial profiles assayed against 39 bacterial indicators (including food-borne and fish pathogenic bacteria in aquaculture as well as other spoilage bacteria) showed that 46% of the isolates exhibited a large inhibition spectrum mainly towards Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Carnobacterium strains. Therefore, highly inhibitory enterococcal strains could potentially be used as probiotics in sea bream and other farming fish fields. Key words: Enterococcus, lactic acid bacteria, aquaculture, probiotic, sea bream.

Spoilage-Related Activity of Carnobacterium maltaromaticum Strains in Air-Stored and Vacuum-Packed Meat

One hundred three isolates of Carnobacterium spp. from raw meat were analyzed by random amplification of polymorphic DNA (RAPD) and PCR and were identified by 16S rRNA gene sequencing. Forty-five strains of Carnobacterium maltaromaticum were characterized for their growth capabilities at different temperatures, NaCl concentrations, and pH values and for in vitro lipolytic and proteolytic activities. Moreover, their spoilage potential in meat was investigated by analyzing the release of volatile organic compounds (VOCs) in meat stored in air or vacuum packs. Almost all the strains were able to grow at 4, 10, and 20°C, at pH values of 6 to 9, and in the presence of 2.5% NaCl. The release of VOCs by each strain in beef stored at 4°C in air and vacuum packs was evaluated by headspace solid-phase microextraction (HS-SPME)-gas chromatography-mass spectrometry (GC-MS) analysis. All the meat samples inoculated and stored in air showed higher numbers of VOCs than the vacuum-packed meat samples. Acetoin, 1-octen-3-ol, and butanoic acid were the compounds most frequently found under both storage conditions. The contaminated meat samples were evaluated by a sensory panel; the results indicated that for all sensory odors, no effect of strain was significant (P > 0.05). The storage conditions significantly affected (P < 0.05) the perception of dairy, spoiled-meat, and mozzarella cheese odors, which were more intense in meat stored in air than in vacuum packs but were never very intense. In conclusion, different strains of C. maltaromaticum can grow efficiently in meat stored at low temperatures both in air and in vacuum packs, producing volatile molecules with low sensory impacts, with a negligible contribution to meat spoilage overall.

Effect of inoculation of Carnobacterium divergens V41, a biopreservative strain against Listeria monocytogenes risk, on the microbiological, chemical and sensory quality of cold-smoked salmon

The aim of this study was to develop a biopreservation strategy for cold-smoked salmon (CSS) by the use of lactic acid bacteria previously selected for their capability to inhibit the growth of Listeria monocytogenes in the product. The spoiling potential of three Carnobacterium strains ( Carnobacterium divergens V41, Carnobacterium piscicola V1 and SF668) was tested in sterile CSS blocks inoculated by 10 4–5 CFU g − 1 and stored under vacuum for 9 days at 4 °C followed by 19 days at 8 °C. C. divergens V41 grew a little faster than the other strains and none of the three carnobacteria showed any adverse effect on quality of the product, i.e. no off-odour detected by a trained panel, no total volatile basic nitrogen (TVBN) production, no acidification and no biogenic amine except a slight production of tyramine. An application on commercial CSS was tested by spraying C. divergens V41 (10 4–5 CFU g − 1 ) on slices of four batches freshly processed in different smokehouses. Microbial, chemical and sensory characteristics were weekly compared to a control during 4 weeks of vacuum storage. When the natural microflora was initially weak (two batches < 20 CFU g − 1 ), C. divergens V41 quickly reached 10 7–8 CFU g − 1 and a slight inhibition of endogenous Enterobacteriaceae, lactobacilli and yeasts was observed. The presence of C. divergens V41 was slightly detected (odour and flavour) but none of the sample was considered as spoiled by the sensory panel. When the natural microflora was initially high (2 batches > 10 4–5 CFU g − 1 ), no effect on the microflora, TVBN and biogenic amine production, nor on the sensory characteristics was observed in presence of C. divergens V41. In conclusion, biopreservation of CSS using lactic acid bacteria such as C. divergens V41 is a promising way to inhibit the growth of pathogenic bacteria such as L. monocytogenes with low effect on the quality of the product.

Quantitative polymerase chain reaction used for the rapid detection of Carnobacterium species from French soft cheeses

An identification method by PCR, specific to the Carnobacterium genus, was optimised by testing it on 28 bacterial strains. Primers from the literature were tested to differentiate Carnobacterium strains present among various bacterial species. The DNA of Carnobacterium species ( C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. viridans), specifically amplified by the Cb1–Cb2R primer couple at a hybridization temperature of 69 °C, gave only one band of 340 bp. The validation of this technique was carried out on a French soft cheese made with pasteurised milk inoculated with C. maltaromaticum LMA 28. Using classical PCR, detection was not possible for decimal dilutions of the cheese above 1 g L −1. With Sybr Green I real time PCR, the specificity of the reaction was confirmed by the T m value. The standard curve constructed using the main threshold cycle and various concentrations of C. maltaromaticum LMA 28 (ranging from 10 0 to 10 8 cfu mL −1) showed good linearity and a sensitivity limit of ⩾10 4 cfu g −1 of cheese. This technique was applied on commercially available cheeses made from raw cow’s milk. The Sybr Green I real time PCR method constitutes an effective and easy-to-perform method to quantify Carnobacterium sp. in cheese.

Biodiversity of Listeria monocytogenes sensitivity to bacteriocin-producing Carnobacterium strains and application in sterile cold-smoked salmon.

The aim of this study was to demonstrate the inhibitory capacity of Carnobacterium strains against a collection of Listeria monocytogenes strains in cold-smoked salmon (CSS). Three bacteriocin-producing strains, Carnobacterium divergens V41, C. piscicola V1 and C. piscicola SF668, were screened for their antilisterial activity against a collection of 57 L. monocytogenes strains selected from the French smoked salmon industry, using an agar spot test. All the Listeria strains were inhibited but three different groups could be distinguished differing in sensitivity to the three Carnobacterium strains. However, C. divergens V41 always had the highest inhibitory effect. The antilisterial capacity was then tested in sterile CSS blocks co-inoculated with Carnobacterium spp. and mixtures of L. monocytogenes strains. C. divergens V41 was the most efficient strain, maintaining the level of L. monocytogenes at <50 CFU g(-1) during the 4 weeks of vacuum storage at 4 and 8 degrees C, whatever the sensitivity of the set of L. monocytogenes strains. C. divergens V41 may be a good candidate for biopreservation in CSS. A biopreservation strategy for CSS against the risk of L. monocytogenes was investigated using bacteriocin-producing lactic acid bacteria.

Whole-organism Fingerprinting of the Genus Carnobacterium using Fourier Transform Infrared Spectroscopy (FT-IR)

Sixty seven strains of Carnobacterium, atypical Lactobacillus, Enterococcus durans, Lactobacillus maltaromicus and Vagacoccus salmoninarum were examined by Fourier transform infrared (FT-IR) spectroscopy. The effects of culture age and reproducibility over a six month period were also investigated. The results were analysed by multivariate statistics and compared with those from a previous numerical phenetic study, a pyrolysis mass spectrometry (PyMS) study and with investigations which used DNA-DNA and 16S rRNA sequencing homologies. Taxonomic correlations were observed between the FT-IR data and these studies. Culture age was observed to have little effect on the spectra obtained. The reproducibility study indicated that there was correlation between spectra produced on two occasions over the six month period. It was concluded that FTIR is a reliable method for investigating carnobacterial classification, and may have further potential as a rapid method for use in Carnobacterium identification.

Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef

Drip samples were collected at 4-week intervals from 10 vacuum-packaged beef striploins stored for 16 weeks at −1.5 °C and assayed for populations of lactic-acid bacteria (LAB), pH and spoilage-causing fermentation products. A total of 15 LAB species were identified using pulsed-field gel electrophoresis and biochemical analysis. A pattern of succession was observed during storage between strains of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. Acetic acid production was associated with increasing LAB populations generally and butyric acid production was associated with the development of a particular strain of Leuconostoc. Changes in pH is postulated as a driver of succession.

Development of genus/species-specific PCR analysis for identification of Carnobacterium strains.

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S-23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes.

The differentiation of Carnobacterium divergens using the random amplification of polymorphic DNA polymerase chain reaction technique.

The potential of the randomly amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) technique to differentiate Carnobacterium divergens from other members of the genus Carnobacterium was examined. A numerical analysis of the genomic profiles obtained demonstrated that it was possible to differentiate the C. divergens strains from other Carnobacterium strains using this technique. The heterogeneity observed in the representatives of the species C. piscicola adds further weight to the suggestion in other taxonomic studies that subspecies of this species exist.

Inhibition of Listeria monocytogenes by In Situ Produced and Semipurified Bacteriocins of Carnobacterium spp. on Vacuum-Packed, Refrigerated Cold-Smoked Salmon

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4°C and 8°C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8°C and had a bacteriostatic effect at 4°C. Listeria growth was not affected by a non–bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4°C and 8°C. Listeria growth was delayed by divercin V41 at 8°C and was inhibited at 4°C. Nisin delayed Listeria growth at 8°C and was bacteriostatic at 4°C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping

A total of 405 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged, salted, sodium nitrite- or potassium nitrate-treated, cold-smoked rainbow trout stored at 4°C or 8°C were characterised and identified using a molecular method. The isolates were initially classified according to their restriction endonuclease profiles using HindIII and EcoRI restriction endonucleases and further characterised by rRNA gene restriction patterns (ribotypes). Numerical analysis of these ribopatterns was performed together with 19 reference LAB strain patterns in order to identify the isolates to species level. The strains were divided with HindIII and EcoRI ribopatterns into ten and nine clusters at the similarity level of 65% and 50%, respectively. The Leuconostoc-clusters and the Lb. sakei/Lb. curvatus-clusters formed the two main groups. Only one isolate was identified as Lactobacillus plantarum and no Carnobacterium strains were discovered. For both enzymes, the 35 isolates possessing six individual ribotypes and forming five clusters could not be identified further with the reference strains used. The relative proportion of Leuconostoc mesenteroides subsp. mesenteroides was higher in all samples stored at 4°C. Most of the Leuconostoc citreum were found in the samples stored at 8°C, and particularly in the nitrite-treated samples.

Inhibition of Listeria monocytogenes by Carnobacterium spp. strains in a simulated cold smoked fish system stored at 4°C

Preservation of smoked salmon from bacterial spoilage, and especially from Listeria monocytogenes, by bacteriocin producers is a promising challenge. Over a hundred lactic acid bacteria, isolated from commercial vacuum packaged cold smoked salmon, were screened for their antagonistic activity against L. innocua. Twenty-two strains were able to produce bacteriocin-like proteinaceous substances. These strains were characterized physiologically and biochemically as Carnobacterium strains. Three different groups were determined by pulsed-field gel electrophoresis after Sma I and Apa I DNA digestion. Peptidoglycan hydrolases patterns completed the characterization of these strains. All were confirmed as being Carnobacterium piscicola. Growth and bacteriocin production of three strains of each group and two well known bacteriocin producers ( C. divergens V41 and C. piscicola V1) were tested in a simulated cold smoked fish system at 4°C. These strains were able to reach 10 8 cfu ml −1 in 21 days and to produce as much bacteriocin activities in the cold smoked fish system as in the rich media. Carnobacterium divergens V41 and C. piscicola V1 were the most effective strains in co-culture experiments, inhibiting L. monocytogenes as early as day 4, whereas C. piscicola SF668 inhibiting effect was observed at day 13. The potential for using such biopreservation treatments on whole smoked salmon is discussed.