Production of Acylated Homoserine Lactones by Psychrotrophic Members of the Enterobacteriaceae Isolated from Foods

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5°C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10⁶ CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5°C under an N₂ atmosphere when mean cell densities increased to 10⁶ CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10⁶ CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

The effect of biogenic amine production by single bacterial cultures and metabiosis on cold-smoked salmon

AIM: Biogenic amines are important indicators of spoilage in vacuum-packed cold-smoked salmon. It is the aim of this study to identify bacteria responsible for biogenic amine production in cold-smoked salmon. METHODS AND RESULTS: The present study identified spoilage microflora from cold-smoked salmon and determined biogenic amine production of single and co-cultures growing in cold-smoked salmon. Photobacterium phosphoreum was the only species that produced histamine when inoculated on sterile cold-smoked salmon. Production of putrescine was enhanced 10-15 times when cultures of Serratia liquefaciens or Hafnia alvei were grown with Carnobacterium divergens or Lactobacillus sakei subsp. carnosus. This phenomenon was explained by interspecies microbial metabolism of arginine, i.e., metabiosis. CONCLUSIONS: The amounts of biogenic amines produced by single and co-cultures corresponded to those observed during spoilage of naturally-contaminated cold-smoked salmon. Photobacterium phosphoreum and Lact. curvatus were identified as the specific spoilage organisms in cold-smoked salmon. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the specific spoilage organism is needed before a model can be developed for shelf-life predictions of cold-smoked salmon.     

Prior frozen storage enhances the effect of edible coatings against Listeria monocytogenes on cold-smoked salmon during subsequent refrigerated storage

Aims: Listeria monocytogenes is a major safety concern for ready‐to‐eat foods. The overall objective of this study was to investigate whether prior frozen storage could enhance the efficacy of edible coatings against L. monocytogenes on cold‐smoked salmon during subsequent refrigerated storage. Methods and Results: A formulation consisting of sodium lactate (SL, 1·2–2·4%) and sodium diacetate (SD, 0·125–0·25%) or 2·5% Opti.Form (a commercial formulation of SL and SD) was incorporated into each of five edible coatings: alginate, κ‐carrageenan, pectin, gelatin and starch. The coatings were applied onto the surface of cold‐smoked salmon slices inoculated with L. monocytogenes at a level of 500 CFU cm^?2. In the first phase, the slices were first frozen at ?18°C for 6 days and stored at 22°C for 6 days. Alginate, gelatin and starch appeared to be the most effective carriers. In the second phase, cold‐smoked salmon slices were inoculated with L. monocytogenes, coated with alginate, gelatin or starch with or without the antimicrobials and stored frozen at ?18°C for 12 months. Every 2 months, samples were removed from the freezer and kept at 4°C for 30 days. Prior frozen storage at ?18°C substantially enhanced the antilisterial efficacy of the edible coatings with or without antimicrobials during the subsequent refrigerated storage. Conclusions: Plain coatings with ≥2 months frozen storage and antimicrobial edible coatings represent an effective intervention to inhibit the growth of L. monocytogenes on cold‐smoked salmon. Significance and Impact of the Study: This study demonstrates the effectiveness of the conjunct application of frozen storage and edible coatings to control the growth of L. monocytogenes to enhance the microbiological safety of cold‐smoked salmon.     

Reduction in Listeria monocytogenes and spoilage bacteria on smoked catfish using X-ray treatments

Aims: To determine the efficacy of X‐ray processes in inactivating L. monocytogenes levels in smoked catfish during storage at 5°C and to determine the effects of X‐ray doses on controlling the growth of spoilage bacteria on smoked catfish during storage at 5°C for up to 5 weeks. Methods and Results: Smoked catfish fillets inoculated with L. monocytogenes were treated with 0·0–2·0 kGy X‐ray and stored at 5°C for 5 weeks. The negative controls (uninoculated/untreated) and uninoculated samples treated with the lowest (0·1 kGy) and highest (2·0 kGy) doses were stored at 5°C and tested for psychrotrophs count during the 5 weeks of storage. The initial L. monocytogenes population on smoked catfish was significantly (P < 0·05) reduced to undetectable level by a treatment of 1·0 kGy or higher. The initial psychrotrophs count on smoked catfish was significantly reduced from 4·7 CFU g^?1 to below the detectable level by a treatment with 2·0 kGy. Conclusions: Smoked catfish treated with 2·0 kGy X‐ray had no detectable L. monocytogenes throughout 35 days of storage at 5°C. A treatment with 2·0 kGy X‐ray also kept the levels of psychrotrophs in the smoked catfish within the acceptable level until 35 days. Significance and Impact of the Study: The results of this investigation indicate that X‐ray at 2·0 kGy can eliminate L. monocytogenes and extend the shelf life of smoked catfish stored at refrigeration temperature.     

The growth potential of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes in dairy manure‐based compost in a greenhouse setting under different seasons

Aim: The pathogen growth in dairy compost was studied in a greenhouse setting under different seasons. Methods and Results: The five‐strain mixtures of each Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes were inoculated separately into dry compost to yield c. 1 log CFU g^?1. After acclimation at room temperature, the inoculated compost was initially adjusted to moisture levels of 10–50% and then kept in a greenhouse under different seasons. The populations of all three pathogens increased by 2·1–3·9 log CFU g^?1 within 3 days in autoclaved compost with initial moisture content of at least 40%. Listeria monocytogenes multiplied up to 2·4 log CFU g^?1 in compost with initial moisture content of 30% and was detected up to 28 days for all seasons, whereas populations of both E. coli O157:H7 and Salmonella increased by c. 1 log in compost with initial moisture content of 30% during winter months only. No pathogen growth in nonautoclaved compost was detected. Conclusion: Bacterial species, temperature, light intensity and moisture content affected the growth potential and survival of pathogens in compost when the population of background microflora was low. Significance and Impact of the Study: Keeping compost as dry as possible and maintaining certain levels of background microflora may be critical to prevent the growth of pathogens.     

Antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food

Aim: To develop antibody–aptamer functionalized fibre‐optic biosensor for specific detection of Listeria monocytogenes from food products. Methods and Results: Aptamer, a single‐stranded oligonucleotide ligand that displays affinity for the target molecule, was used in the assay to provide sensor specificity. Aptamer‐A8, specific for internalin A, an invasin protein of L. monocytogenes, was used in the fibre‐optic sensor together with antibody in a sandwich format for detection of L. monocytogenes from food. Biotinylated polyclonal anti‐Listeria antibody, P66, was immobilized on streptavidin‐coated optical waveguide surface for capturing bacteria, and Alexa Fluor 647‐conjugated A8 was used as a reporter. The biosensor was able to selectively detect pathogenic Listeria in pure culture and in mixture with other bacteria at a concentration of approx. 10³ CFU ml^?1. This sensor also successfully detected L. monocytogenes cells from artificially contaminated (initial inoculation of 10² CFU 25 g^?1) ready‐to‐eat meat products such as sliced beef, chicken and turkey after 18 h of enrichment. Conclusion: Based on the data presented in this study, the antibody–aptamer functionalized fibre‐optic biosensor could be used as a detection tool for sensitive and specific detection of L. monocytogenes from foods. Significance and Impact of the Study: The study demonstrates feasibility and novel application of aptamer on fibre‐optic biosensor platform for the sensitive detection of L. monocytogenes from food products.     

Production of acylated homoserine lactones by psychrotrophic members of the Enterobacteriaceae isolated from foods.

Bacteria are able to communicate and gene regulation can be mediated through the production of acylated homoserine lactone (AHL) signal molecules. These signals play important roles in several pathogenic and symbiotic bacteria. The following study was undertaken to investigate whether AHLs are produced by bacteria found in food at temperatures and NaCl conditions commercially used for food preservation and storage. A minimum of 116 of 154 psychrotrophic Enterobacteriaceae strains isolated from cold-smoked salmon or vacuum-packed chilled meat produced AHLs. Analysis by thin-layer chromatography indicated that N-3-oxo-hexanoyl homoserine lactone was the major AHL of several of the strains isolated from cold-smoked salmon and meat. AHL-positive strains cultured at 5 degrees C in medium supplemented with 4% NaCl produced detectable amounts of AHL(s) at cell densities of 10(6) CFU/ml. AHLs were detected in cold-smoked salmon inoculated with strains of Enterobacteriaceae stored at 5 degrees C under an N(2) atmosphere when mean cell densities increased to 10(6) CFU/g and above. Similarly, AHLs were detected in uninoculated samples of commercially produced cold-smoked salmon when the level of indigenous Enterobacteriaceae reached 10(6) CFU/g. This level of Enterobacteriaceae is often found in lightly preserved foods, and AHL-mediated gene regulation may play a role in bacteria associated with food spoilage or food toxicity.     

Comparison of Campylobacter jejuni isolates from human, food, veterinary and environmental sources in Iceland using PFGE, MLST and fla-SVR sequencing

Aims: To evaluate the probiotic properties of strains isolated from smoked salmon and previously identified as bacteriocin producers. Methods and Results: Strains Lactobacillus curvatus ET06, ET30 and ET31, Lactobacillus fermentum ET35, Lactobacillus delbrueckii ET32, Pediococcus acidilactici ET34 and Enterococcus faecium ET05, ET12 and ET88 survived conditions simulating the gastrointestinal tract (GIT) and produced bacteriocins active against several strains of Listeria monocytogenes, but presented very low activity against other lactic acid bacteria (LAB). Cell‐free supernatants containing bacteriocins, added to 3‐h‐old cultures of L. monocytogenes 603, suppressed growth over 12 h. Auto‐aggregation was strain‐specific, and values ranged from 7·2% for ET35 to 12·1% for ET05. Various degrees of co‐aggregation with L. monocytogenes 603, Lactobacillus sakei ATCC 15521 and Enterococcus faecalis ATCC 19443 were observed. Adherence of the bacteriocinogenic strains to Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. The highest levels of hydrophobicity were recorded for Lact. curvatus (61·9–64·6%), Lact. fermentum (78·9%), Lact. delbrueckii (43·7%) and Ped. acidilactici (51·3%), which are higher than the one recorded for Lact. rhamnosus GG (53·3%). These strains were highly sensitive to several antibiotics and affected by several drugs from different generic groups in a strain‐dependent manner. Conclusions: Smoked salmon is a rich source of probiotic LAB. All strains survived conditions simulating the GIT and produced bacteriocins active against various pathogens. Adherence to Caco‐2 cells was within the range reported for Lact. rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Smoked salmon contains a number of different probiotic LAB and could be marketed as having a potential beneficial effect.     

Ascomycetous yeast species recovered from grapes damaged by honeydew and sour rot

Aims: To identify ascomycetous yeasts recovered from sound and damaged grapes by the presence of honeydew or sour rot. Methods and Results: In sound grapes, the mean yeast counts ranged from 3·20 ± 1·04 log CFU g^?1 to 5·87 ± 0·64 log CFU g^?1. In honeydew grapes, the mean counts ranged from 3·88 ± 0·80 log CFU g^?1 to 6·64 ± 0·77 log CFU g^?1. In sour rot grapes counts varied between 6·34 ± 1·03 and 7·68 ± 0·38 logCFU g^?1. Hanseniaspora uvarum was the most frequent species from sound samples. In both types of damage, the most frequent species were Candida vanderwaltii, H. uvarum and Zygoascus hellenicus. The latter species was recovered in high frequency because of the utilization of the selective medium DBDM (Dekkera/Brettanomyces differential medium). The scarce isolation frequency of the wine spoilage species Zygosaccharomyces bailii (in sour rotten grapes) and Zygosaccharomyces bisporus (in honeydew affected grapes) could only be demonstrated by the use of the selective medium ZDM (Zygosaccharomyces differential medium). Conclusions: The isolation of several species only from damaged grapes indicates that damage constituted the main factor determining yeast diversity. The utilization of selective media is required for eliciting the recovery of potentially wine spoilage species. Significance and Impact of the Study: The impact of damaged grapes in the yeast ecology of grapes has been underestimated.     

Lactic acid bacteria with potential to eliminate fungal spoilage in foods

Aims: To investigate antifungal activity produced by lactic acid bacteria (LAB) isolated from malted cereals and to determine if such LAB have the capacity to prevent fungal growth in a particular food model system. Methods and Results: The effect of pH, temperature and carbon source on production of antifungal activity by four LAB was determined. Pediococcus pentosaceus was used to conduct a trial to determine if it is feasible to eliminate Penicillium expansum, the mould responsible for apple rot, using an apple model. Penicillium expansum was incapable of growth during the trial on apple‐based agar plates inoculated with the antifungal‐producing culture, whereas the mould did grow on apple plates inoculated with an LAB possessing no antifungal activity. Conclusion: Partial characterization of the antifungal compounds indicates that their activity is likely to be because of production of antifungal peptides. The trial conducted showed that the antifungal culture has the ability to prevent growth of the mould involved in apple spoilage, using apples as a model. Significance and Impact of the study: The ability of an LAB to prevent growth of Pen. expansum using the apple model suggests that these antifungal LAB have potential applications in the food industry to prevent fungal spoilage of food.     

Pulsed electric field treatment as a potential method for microbial inactivation in scaffold materials for tissue engineering: the inactivation of bacteria in collagen gel

Aims: To investigate the effectiveness of pulsed electric field (PEF) treatment as a new method for inactivation of micro-organisms in complex biomatrices and to assess this by quantifying the inactivation of Escherichia coli seeded in collagen gels. Methods and Results: PEF was applied to E. coli seeded collagen gels in static (nonflowing) chambers. The influence of electric field strength, pulse number and seeded cell densities were investigated. The highest level of inactivation was obtained at the maximum field strength of 45 kV cm⁻¹. For low levels of E. coli contamination (10³ CFU ml⁻¹), PEF treatment resulted in no viable E. coli being recovered from the gels. However, PEF treatment of gels containing higher cell densities (≥10⁴ CFU ml⁻¹) did not achieve complete inactivation of E. coli. Conclusions: PEF treatment successfully inactivated E. coli seeded in collagen gels by 3 log₁₀ CFU ml⁻¹. Complete inactivation was hindered at high cell densities by the tailing effect observed. Significance and Impact of the Study: PEF shows potential as a novel, nondestructive method for decontamination of collagen-based matrices. Further investigation is required to ensure its compatibility with other proteins and therapeutic drugs for tissue engineering and drug delivery applications.     

Characterization of a broad range antibacterial substance from a new <Emphasis Type="Italic">Bacillus</Emphasis> species isolated from Amazon basin

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, incluing pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml⁻¹. The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Inhibition of Listeria monocytogenes in Cooked Ham by Virulent Bacteriophages and Protective Cultures

Protective cultures can be used successfully as an additional hurdle together with phages to reduce growth of Listeria monocytogenes on sliced cooked ham. Addition of phages resulted in a rapid 10-fold reduction of L. monocytogenes. After 14 to 28 days of storage, a 100-fold reduction was observed in samples with phages and protective culture compared to results for samples with phages alone.Listeriosis in Europe has an average incidence between 2 and 10 reported cases per million population per year (7). Listeria monocytogenes is found in raw and ready-to-eat (RTE) products, poultry, seafood, and dairy products. A review of the incidence and transmission of L. monocytogenes in RTE products has been published by Lianou and Sofos (11). The USDA has implemented a “zero-tolerance” policy for L. monocytogenes in RTE products (2). In the European Union, the limit for common RTE foods is 100 CFU/g (1). Recently, Codex Alimentarius adopted new standards for L. monocytogenes in RTE foods, with a limit of 100 CFU/g in foods where L. monocytogenes cannot grow and absence in foods where the bacterium can grow. However, an alternative approach is accepted. Competent authorities may choose to establish and implement other validated limits (http://www.codexalimentarius.net/web/archives.jsp?lang=en, Alinorm 09/32/REP and Alinorm 09/32/13). L. monocytogenes in cooked products is connected with cross-contamination after heat treatment (11, 12). Bacteriophages have been successfully applied to a number of food products to reduce the level of contaminating L. monocytogenes (6, 8-10, 13, 14). The effect of phages varies with the type of product and is strongly dose dependent (6, 8). Active phages can be recovered from foods after long storage, but the phage particles appear to become immobilized soon after addition to nonliquid foods and therefore, due to limited diffusion, cannot infect bacteria (8). Bacteria surviving phage treatment can later grow in the product. Additional hurdles should therefore be present to inhibit later outgrowth of L. monocytogenes.We have previously employed Lactobacillus sakei TH1 as a protective culture against L. monocytogenes (4, 5). Here we examine the combined use of phages and protective culture to reduce outgrowth of L. monocytogenes on cooked ham.Rifampin (rifampicin)-resistant mutants of L. monocytogenes 2230/92 serotype 1, implicated in a listeriosis outbreak in Norway (12), and L. monocytogenes 167 serotype 4b were grown overnight in brain heart infusion (Difco Laboratories, Detroit, MI) at 37°C without shaking and stored at 4°C for 24 h (3-5). Cells were diluted in 0.9% NaCl and plated on brain heart infusion agar with 200 μg/ml rifampin. L. sakei TH1 was grown at 30°C in MRS (de Man, Rogosa, Sharpe) broth (CM 359; Oxoid, Hampshire, England) (pH 6.2) and plated on MRS agar (5). Listex P100 phages, 2 × 10¹¹ PFU/ml, were from EBI Food Safety (Wageningen, The Netherlands).Ten-gram slices of hams with 2.3% NaCl and 0.01% disodium diphosphate (pH 6.2; a_w > 0.97), made at Nofima''s pilot plant (Aas, Norway), were inoculated with a cold-adapted 1:1 mixture of L. monocytogenes 2230/92 and 167. Bacteria were spread in 100 μl 0.9% NaCl over the 80-cm² surface area of each slice to 10³ CFU/cm² using a bent glass rod. After 1 h at 20°C, phages (5 × 10⁷ PFU/cm² in a total volume of 100 μl) were spread over the same surface. After one additional hour, 10³ CFU/cm² L. sakei TH1 in 100 μl 0.9% NaCl was added where appropriate. The slices were vacuum packed and stored at 10°C. Growth was measured before and after spiking and at 0, 3, 7, 14, and 28 days after homogenizing the slices in 100 ml 0.9% NaCl in a Stomacher homogenizer. No lactic acid bacteria were detected in uninoculated samples. Experiments were performed with three parallel samples. L. monocytogenes alone grew from 10⁴ CFU/g at the onset of the experiment to 10⁷ CFU/g the first 7 days, reached 2 × 10⁸ CFU/g after 14 days, and remained unchanged thereafter (Fig. ​(Fig.1).1). In samples with both L. monocytogenes and phages, a rapid 1-log reduction in L. monocytogenes was observed. Surviving L. monocytogenes, however, grew as well as that in the phage-free controls, reaching >10⁷ CFU/g after 14 days. In samples where both P100 phages and L. sakei TH1 were added, the same initial reduction of L. monocytogenes was observed, but the later outgrowth was reduced by the fast-growing lactic acid bacteria and the L. monocytogenes levels were 2 logs lower than those with P100 phages alone after 28 days of incubation. The phages did not influence the growth and survival of L. sakei TH1. During the 28 days of storage, the pH changed from 6.20 to 6.05 in samples with L. monocytogenes and to 6.00 in samples with both L. monocytogenes and L. sakei TH1. The results were reproduced in a separate repetition of the experiment at 10°C (not shown).Open in a separate windowFIG. 1.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 5 × 10⁷ PFU/cm² P100 phages (▪), or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10³ CFU/cm² (approximately 10⁴ CFU/g) protective-culture L. sakei TH1 (▴) and stored at 10°C. Growth of L. sakei TH1 is shown by the broken line.The effect of the protective culture was dose dependent when 10⁴ CFU/g and 10⁶ CFU/g of L. sakei TH1 were added to slices of ham (Fig. ​(Fig.2).2). L. monocytogenes alone grew to 2 × 10⁸ CFU/g after 14 days. When L. sakei TH1 was added at a low concentration (10⁴ CFU/g), L. monocytogenes grew to approximately 4 × 10⁶ CFU/g, while when L. sakei TH1 was added at a high concentration, L. monocytogenes levels were 1 to 2 logs lower. The pHs in the low- and high-inoculum hams were reduced from the initial 6.20 to 6.16 and 6.02, respectively, at day 28. For hams stored at 4°C, slow growth of L. monocytogenes occurred between days 14 and 28 from 10⁴ to 10⁵ CFU/g (P = 0.003) (Fig. ​(Fig.3).3). With phages and L. sakei TH1 added, a rapid 1-log reduction of L. monocytogenes was observed due to the phage attack, and no growth was observed during the 28-day storage period. The L. sakei TH1 strain showed a longer lag phase at this low temperature but nevertheless reached 10⁷ CFU/g at day 14 and thereby inhibited any growth of L. monocytogenes.Open in a separate windowFIG. 2.Inhibition of L. monocytogenes in cooked ham inoculated with large or small amounts of protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 10⁶ CFU/g (10⁵ CFU/cm²) L. sakei TH1 (▴), or L. monocytogenes and 10⁴ CFU/g (10³ CFU/cm²) L. sakei TH1 (▪) and stored at 10°C. Growth of L. sakei TH1 is shown by broken lines. The L. monocytogenes control is the same control as in Fig. ​Fig.11.Open in a separate windowFIG. 3.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 4°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫) or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10⁴ CFU/g (10³ CFU/cm²) protective-culture, L. sakei TH1 (▴) and stored at 4°C. Growth of L. sakei TH1 is shown by the broken line.Since L. sakei TH1 grows well at low temperatures, prevents growth of L. monocytogenes, and has no negative influence on the organoleptic properties of ham (4, 5), it can successfully be employed as an additional hurdle together with phages.We here chose to perform the storage experiments under “worst-case” conditions. Generally, the contamination levels of L. monocytogenes are lower than in our setup, in the range of 10 to 100 CFU/g (see reference 11 and references therein). Since L. sakei TH1 grows well at low temperatures (Fig. ​(Fig.3),3), its selective advantage will be greater at 4°C than at abuse temperatures. From the above, it is evident that it is possible to optimize L. monocytogenes inhibition by increasing both the phage titer and the starting amount of protective culture. An enhanced effect may also be experienced by modifying phage application, e.g., by using larger liquid volumes (6, 8).Emergence of resistant L. monocytogenes may be a potential problem when treating foods with phages. No emergence of resistance has been detected after phage treatment (6, 8). Such strategies as use of phage mixtures, phage rotation schemes, and treatment of products immediately prior to packaging may reduce eventual resistance problems (8). Some L. monocytogenes strains are naturally phage resistant (6). In these cases, a protective culture still constitutes a powerful hurdle.In conclusion, we have shown here that by applying phages and protective culture as two independent hurdles, it is possible to both reduce the number of L. monocytogenes bacteria on a product and inhibit outgrowth of eventual remaining surviving cells. This is a general method that can potentially be applied to different foods where there is a potential risk for growth of L. monocytogenes, provided a suitable protective culture is available.     

The bacterial flora of vacuum-packed cold-smoked salmon stored at 7 degrees C, identified by direct 16S rRNA gene analysis and pure culture technique

AIMS: The indigenous flora of freshly chilled cold-smoked salmon just after the vacuum packaging, and the spoilage flora after storage, in vacuum package at 7 degrees C for 19 days, were to be investigated with two different sampling strategies. METHODS AND RESULTS: Identification was performed using 16S rRNA sequencing of both isolated bacteria and bacterial DNA from tissue extract. The indigenous flora of fresh cold-smoked vacuum-packed salmon was dominated by, in order, Brochothrix thermosphacta, Yersinia ruckeri, Photobacterium and Carnobacterium, whereas the spoilage flora of the same product stored at 7 degrees C for 19 days was dominated by Lactobacillus and Photobacterium. The two sampling strategies showed similar results on the fish flora. Several new types of Photobacterium sequences, closely related to Photobacterium iliopiscarium and Photobacterium phosphoreum, were found from both the freshly processed and the stored salmon, indicating that smoked salmon harbours at least three different, as yet unknown, Photobacterium species. CONCLUSIONS: Ten per cent of the bacterial flora multiplying on chilled, vacuum-packed, cold-smoked salmon comprised unknown species. The two sampling strategies complement each other. SIGNIFICANCE AND IMPACT OF THE STUDY: As cold-smoked salmon is consumed without heat-treatment, the presence of undefined bacteria in high numbers should be considered in public health assessments.     

Inhibition of Brochothrix thermosphacta and sensory improvement of tropical peeled cooked shrimp by Lactococcus piscium CNCM I‐4031

Aims: To investigate the antimicrobial spectrum of Lactococcus piscium CNCM I‐4031 and its protective effect in cooked and peeled shrimp against Brochothrix thermosphacta. Methods and Results: Sixteen pathogenic and spoiling bacteria were inhibited in Elliker, but not in shrimp juice agar plates. In shrimp packed under modified atmosphere and stored at 8°C, B. thermosphacta (10³ CFU g^?1) was inhibited by 4·1 log CFU g^?1 when co‐inoculated with L. piscium (10⁶ CFU g^?1). Brochothrix thermosphacta spoiled the product after 11 days, with the emission of strong butter/caramel off‐odours. In co‐culture with L. piscium, sensory shelf‐life was extended by at least 10 days. The inhibition was partially explained by a drop in pH from 6·6 to 5·6. The physicochemical composition of shrimp and shrimp juice was established to identify the inhibition mechanisms involved. Conclusion: Lactococcus piscium CNCM I‐4031 has a wide antimicrobial spectrum. The strain inhibits B. thermosphacta in shrimp and significantly prolongs sensory shelf‐life. Significance and Impact of the Study: Lactococcus piscium CNCM I‐4031 is shown to be a promising agent for improving shrimp quality and may be tested against pathogens and in other food matrices. Knowledge of the physicochemical composition of shrimp and shrimp juice will allow the development of a chemically defined model medium for determining the inhibition mechanisms involved.     

Nisin‐induced expression of pediocin in dairy lactic acid bacteria

Aims: To test whether a single vector, nisin‐controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0·8‐kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml^?1 nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.     

Elucidation of Listeria monocytogenes Contamination Routes in Cold-Smoked Salmon Processing Plants Detected by DNA-Based Typing Methods

The contamination routes of Listeria monocytogenes in cold-smoked salmon processing plants were investigated by analyzing 3,585 samples from products (produced in 1995, 1996, 1998, and 1999) and processing environments (samples obtained in 1998 and 1999) of two Danish smokehouses. The level of product contamination in plant I varied from 31 to 85%, and no L. monocytogenes was found on raw fish (30 fish were sampled). In plant II, the levels of both raw fish and product contamination varied from 0 to 25% (16 of 185 raw fish samples and 59 of 1,000 product samples were positive for L. monocytogenes). A total of 429 strains of L. monocytogenes were subsequently compared by random amplified polymorphic DNA (RAPD) profiling, and 55 different RAPD types were found. The RAPD types detected on the products were identical to types found on the processing equipment and in the processing environment, suggesting that contamination of the final product (cold-smoked salmon) in both plants (but primarily in plant I) was due to contamination during processing rather than to contamination from raw fish. However, the possibility that raw fish was an important source of contamination of the processing equipment and environment could not be excluded. Contamination of the product occurred in specific areas (the brining and slicing areas). In plant I, the same RAPD type (RAPD type 12) was found over a 4-year period, indicating that an established in-house flora persisted and was not eliminated by routine hygienic procedures. In plant II, where the prevalence of L. monocytogenes was much lower, no RAPD type persisted over long periods of time, and several different L. monocytogenes RAPD types were isolated. This indicates that persistent strains may be avoided by rigorous cleaning and sanitation; however, due to the ubiquitous nature of the organism, sporadic contamination occurred. A subset of strains was also typed by using pulsed-field gel electrophoresis and amplified fragment length polymorphism profiling, and these methods confirmed the type division obtained by RAPD profiling.