Lisbeth Truelstrup Hansen

Effects of salt and storage temperature on chemical, microbiological and sensory changes in cold-smoked salmon

Abstract Chemical, microbiological and sensory changes during storage of vacuum-packed cold-smoked salmon were studied using a factorial experimental design with two storage temperatures (5 and 10 °C) and two salt levels (2.2 and 4.6%). The spoilage characteristics were typical of microbiological activity in all treatments, but there was no relation between sensory changes and any of the microbiological numbers (total viable counts, total psychrotrophes, lactic acid bacteria or Enterobacteriaceae). Total viable counts typically reached 108 cfu/g weeks before sensory rejection. Acetic acid, hypoxanthine, trimethylamine and ethanol concentrations increased with storage time in all treatments. The increase in ethanol depended on salt concentration but not storage temperature. Absolute values of trimethylamine ranged from 2–8 mg TMA-N/100 g initially, to 10 mg TMA-N/100 g at sensory rejection. Acetic acid levels increased with temperature and decreased with salt concentration, but varied between 12–23 μmol g at rejection. Initial concentrations of hypoxanthine increased from 2–3 μmol g to maximum 8–9 μmol g , with values of 5–7 μmol g indicating the limit of sensory acceptability. Hypoxanthine was considered to be the best objective indicator for sensory quality of cold-smoked salmon.

Importance of autolysis and microbiological activity on quality of cold-smoked salmon

The relative importance of autolysis and microbiological activity on spoilage of cold-smoked salmon and the origin of the chemical compounds hypoxanthine, acetic acid, trimethylamine and total volatile bases were studied in a storage experiment of dry salted and injection-brined, vacuum-packed salmon with normal and reduced loads of microorganisms. Comparative studies of cold-smoked salmon with a reduced and normal load of microorganisms showed that microbiological activity caused production of the characteristic spoilage odours and flavours, while the autolytic enzymes from the fish tissue had major impact on the textural deterioration. Total volatile bases and hypoxanthine were produced in significantly higher levels in salmon with a normal bacterial load. Sixty-eight per cent of the hypoxanthine found in stored samples originated from microbiological conversion of inosine to hypoxanthine. The concentration of acetic acid only increased in samples with a normal load. At the onset of spoilage, the microflora in dry salted salmon was dominated by marine vibrio in contrast to the injection brined product where a mixture of Enterobacteriaceae and lactic acid bacteria were prevalent. The different ratios of hypoxanthine produced to trimethylamine also suggested that spoilage of salmon salted by the two methods was caused by different microorganisms.

Comparison of the microflora isolated from spoiled cold-smoked salmon from three smokehouses

Abstract The microflora on spoiled, sliced and vacuum packed, cold-smoked salmon from three smokehouses was quantified and characterized in two independent experiments. Large variations in the microflora were observed both within (i.e. among vacuum packs from the same batch) and among the smokehouses. Lactic acid bacteria dominated the microflora, which reached 107 cfu g−1. Total viable counts of microorganisms alone were not related to quality, though spoilage characteristics were typical for microbiological spoilage. Among the lactic acid bacteria, Lactobacillus curvatus (ca 52–55%) was the most common species in both experiments with Lactobacillus sake, Lactobacillus plantarum, Carnobacterium spp. and Leuconostoc spp. present in smaller numbers. In some cases, large numbers of Enterobacteriaceae were also present and identified species were Serratia liquefaciens, Enterobacter agglomerans and Hafnia alvei. The microflora on cold-smoked salmon appeared to be related to the source of contamination i.e. the raw material and/or the smokehouse rather than being specific for the product, thus rendering the identification of the specific spoilage organisms difficult.

Antibacterial effect of protamine in combination with EDTA and refrigeration

The antimicrobial effect of protamine (clupeine) on a range of gram-positive and gram-negative foodborne pathogens and spoilage bacteria, was evaluated using an agar dilution assay and a broth dilution assay with Alamar Blue as growth indicator. Protamine was tested alone at concentrations from 0 to 10,000 microg/ml, and in combination with EDTA (0.9 mM). Assays were performed at 5 degrees C, 10 degrees C, 18 degrees C and 30 degrees C to test the effect of temperature. Minimum inhibitory concentration (MIC) values ranged from 10 microg/ml for Brochothrix thermosphacta to no inhibition at 10,000 microg/ml for bacteria such as Aeromonas hydrophila, proteolytic strains of Clostridium botulinum, Hafnia alvei and Morganella morganii. The minimum bactericidal concentrations (MBCs) were generally higher than MICs. In combination with EDTA, MICs of protamine decreased for gram-negative test strains, whereas EDTA alone inhibited gram-positive strains. The effect of assay incubation temperature was variable and not clear for most strains. Concentrations of 100-750 microg/ml protamine inhibited the five non-proteolytic C. botulinum strains, while none of the eight proteolytic strains was inhibited, indicating the possible role of proteolytic enzymes in protecting cells from protamine. Clearing zones, indicative of proteolytic activity, were observed in the opaque TSB-agarose around colonies of some but not all protamine-resistant bacteria, suggesting that this is not the only resistance mechanism. Addition of 5% (w/v) gelatin to study the effect of an increased protein concentration in the agar dilution assay showed that electrostatic interactions between protamine and the protein decreased the antimicrobial efficacy of the peptide.

The survival of Listeria monocytogenes during long term desiccation is facilitated by sodium chloride and organic material

One specific DNA-subtype, as determined by RAPD, of Listeria monocytogenes persisted in a fish slaughterhouse for years, even during months with no production where the plant was cleaned and kept dry. We hypothesised that tolerance to desiccation could be a factor in explaining the persistence of L. monocytogenes in food processing environments and the purpose of the present study was to determine ability of L. monocytogenes to survive desiccation on stainless steel under simulated food processing conditions. Viable counts of eight different L. monocytogenes strains exposed to different soils and relative humidities (RHs) during desiccation decreased significantly (p<0.05) during the first week but subsequently remained constant at a plateau for weeks or even months thereafter. Desiccation in physiological peptone saline (PPS) reduced survivors by 3-5 log units whereas bacterial cells suspended in bacteriological growth substrates (tryptone soy broth with 1% glucose, TSB-glu) or PPS with 5% NaCl only were reduced by 1-3 log units. At RHs of 2, 43 and 75%, surfaces were visibly dry after 1, 3 and 5days of incubation, respectively. The lowest RH resulted in the most significant loss of viability, however, 10(3)-10(4)CFU/cm(2) remained viable regardless of the desiccation treatment (i.e., presence of TSB-glu and/or salt). At 75% RH, the bacterial counts remained almost constant when desiccated in TSB-glu. When bacteria were grown and desiccated (15 degrees C, 43% RH) in salmon or smoked salmon juice, survivors decreased slowly resulting in low numbers (10(2)-10(3)CFU/cm(2)) from all eight strains remaining viable after 3months. Whilst conditions during desiccation had a pronounced influence on inactivation kinetics and the number of survivors, persistent L. monocytogenes were not more tolerant to desiccation than presumed non-persistent isolates. Our study shows that the ability to survive for months during desiccated conditions may be a factor explaining the ability of L. monocytogenes to persist in food processing environments.

Desiccation of adhering and biofilm Listeria monocytogenes on stainless steel: Survival and transfer to salmon products.

The foodborne bacterial pathogen, Listeria monocytogenes, commonly contaminates foods during processing, where the microorganisms are potentially subjected to low relative humidity (RH) conditions for extended periods of time. The objective of this study was to examine survival during desiccation (43% RH and 15 °C) of biofilm L. monocytogenes N53-1 cells on stainless steel coupons and to assess subsequent transfer to salmon products. Formation of static biofilm (2 days at 100% RH and 15 °C) prior to desiccation for 23 days significantly (P<0.05) improved survival of cells desiccated in initial low salt concentrations (0.5%) compared to the survival for non-biofilm cells also desiccated in low salt, indicating the protective effect of the biofilm matrix. Osmoadaptation of cells in 5% NaCl before formation of the static biofilm significantly (P<0.05) increased long-term desiccation survival (49 days) irrespectively of the initial salt levels (0.5% and 5% NaCl). The efficiency of transfer (EOT) of desiccated biofilm cells was significantly (P<0.05) lower than EOTs for desiccated non-biofilm bacteria, however, as biofilm formation enhanced desiccation survival more bacteria were still transferred to smoked and fresh salmon. In conclusion, the current work shows the protective effect of biofilm formation, salt and osmoadaptation on the desiccation survival of L. monocytogenes, which in turn increases the potential for cross-contamination during food processing.

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens, Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N21 days of Serratia to be ca 3 Log10(CFU cm−2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.

Effect of cold-smoking and drying on the textural properties of farmed Atlantic salmon (Salmo salar).

Abstract Atlantic salmon fillets were brine-salted, chilled and cold smoked for different times to gain knowledge about the effect of smoking and drying on the quality and acceptability of cold-smoked salmon. Textural properties were determined instrumentally and by sensory evaluation. Salt content ranged from 2.2 to 3.6% salt in water phase salt (WPS) with the highest levels in fillets smoked and dried for 8 h. Moisture content varied inversely with salt, and total fat increased with increasing smoking time. Break strength increased significantly with decrease in moisture. Although there was a highly significant correlation between instrumental texture data and sensory data for cohesiveness and bite resistance, the instrumental method was more discriminating. Panelists found significant differences in texture, juiciness and overall acceptability, and preferred samples which were smoked and dried for longer time periods.

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed

ABSTRACT Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.

Increased thermal and osmotic stress resistance in Listeria monocytogenes 568 grown in the presence of trehalose due to inactivation of the phosphotrehalase-encoding gene treA.

ABSTRACT The food-borne pathogen Listeria monocytogenes is a problem for food processors and consumers alike, as the organism is resistant to harsh environmental conditions and inimical barriers implemented to prevent the survival and/or growth of harmful bacteria. One mechanism by which listeriae mediate survival is through the accumulation of compatible solutes, such as proline, betaine and carnitine. In other bacteria, including Escherichia coli, the synthesis and accumulation of another compatible solute, trehalose, are known to aid in the survival of stressed cells. The objective of this research was to investigate trehalose metabolism in L. monocytogenes, where the sugar is thought to be transferred across the cytoplasmic membrane via a specific phosphoenolpyruvate phosphotransferase system and phosphorylation to trehalose-6-phosphate (T6P). The latter is subsequently broken down into glucose and glucose-6-phosphate by α,α-(1,1) phosphotrehalase, the putative product of the treA gene. Here we report on an isogenic treA mutant of L. monocytogenes 568 (568:ΔTreA) which, relative to the wild-type strain, displays increased tolerances to multiple stressors, including heat, high osmolarity, and desiccation. This is the first study to examine the putative trehalose operon in L. monocytogenes, and we demonstrate that lmo1254 (tre A) in L. monocytogenes 568 indeed encodes a phosphotrehalase required for the hydrolysis of T6P. Disruption of the treA gene results in the accumulation of T6P which is subsequently dephosphorylated to trehalose in the cytosol, thereby contributing to the stress hardiness observed in the treA mutant. This study highlights the importance of compatible solutes for microbial survival in adverse environments.