Predicting the concentration of verotoxin-producing Escherichia coli bacteria during processing and storage of fermented raw-meat sausages.

A model to predict the population density of verotoxigenic Escherichia coli (VTEC) throughout the elaboration and storage of fermented raw-meat sausages (FRMS) was developed. Probabilistic and kinetic measurement data sets collected from publicly available resources were completed with new measurements when required and used to quantify the dependence of VTEC growth and inactivation on the temperature, pH, water activity (aw), and concentration of lactic acid. Predictions were compared with observations in VTEC-contaminated FRMS manufactured in a pilot plant. Slight differences in the reduction of VTEC were predicted according to the fermentation temperature, 24 or 34°C, with greater inactivation at the highest temperature. The greatest reduction was observed during storage at high temperatures. A population decrease greater than 6 decimal logarithmic units was observed after 66 days of storage at 25°C, while a reduction of only ca. 1 logarithmic unit was detected at 12°C. The performance of our model and other modeling approaches was evaluated throughout the processing of dry and semidry FRMS. The greatest inactivation of VTEC was predicted in dry FRMS with long drying periods, while the smallest reduction was predicted in semidry FMRS with short drying periods. The model is implemented in a computing tool, E. coli SafeFerment (EcSF), freely available from http://www.ifr.ac.uk/safety/EcoliSafeFerment. EcSF integrates growth, probability of growth, and thermal and nonthermal inactivation models to predict the VTEC concentration throughout FRMS manufacturing and storage under constant or fluctuating environmental conditions.

Microbiota of Atlantic cod (Gadus morhua L.) rearing systems at pre- and posthatch stages and the effect of different treatments.

AIMS: To study the effect of ova disinfection, antibiotic and microbial treatments on the dominant cultivable cod rearing microbiota at pre- and posthatch stages, determining some virulence-related phenotypic traits among bacterial isolates and their relation to larval survival. METHODS AND RESULTS: Sampling of rearing systems (rearing water, ova, larvae, feeds and supplement) for analysis of cultivable microbiota took place at early stages in 2004 and 2005. Cultivation, phenotypic and genotypic (16S rRNA gene) analyses were performed. The production of putative virulence factors (PVFs), including haemolysin, siderophores and quorum-sensing signals, by bacterial isolates was investigated and related to larval survival. The study was performed during two spawning seasons, evaluating current hatchery practices (ova disinfection and antibiotic treatment of unhealthy larvae) and specific putative probiotics applied to ova and larvae or rotifers. A diversified microbiota (75 operational taxonomic units, OTUs) was observed in cod rearing systems influenced by the feeds and treatments, with prevailing γ-Proteobacteria prior to hatching towards a multiphyla microbiota posthatch. Phenotypic tests demonstrated the heterogeneity within some OTUs. Multivariate analysis of survival data in larval silos and the corresponding larval microbiota was used to divide the genotypic groups into beneficial/harmless and detrimental/opportunistic clusters. PVFs were common among the proposed detrimental/opportunistic OTUs. CONCLUSIONS: The results clearly demonstrate the influence of exogeneous feeding and treatments on larval gastrointestinal microbiota and the role of bacteria in larval survival. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased understanding of the microbiota in rearing systems may contribute to successful implementation of microbial management in cod aquaculture.

Effects of bacterial treatment at early stages of Atlantic cod (Gadus morhua L.) on larval survival and development.

AIMS: To assess the effects of bacterial treatment at the earliest stages of cod rearing on the microbial load, larval development and performance, testing three bacterial strains (Carnobacterium divergens V41, Arthrobacter sp. and Enterococcus sp.) in vivo that were previously shown to have inhibitory potential towards fish pathogens in vitro. METHODS AND RESULTS: A bacterial mixture was added eight times to the rearing water from the prehatch to the mid-larval stage (a 38-day period). Microbiological analysis of ova, larvae and rearing water was performed regularly. Larval performance and development were evaluated by survival rate, hypersalinity tolerance and physiological measurements. Different larval survival rates were observed within and between treatments, and possibly explained by variations in larval microflora and established probionts. Larvae from one silo, which had been bathed in the bacterial suspension, showed the highest survival rate (42.1%), lowest Vibrio levels, and were significantly heavier (19.3%) and more stress tolerant than control larvae (P < 0.01). This coincided with the intestinal establishment of two of the tested bacteria. CONCLUSIONS: Arthrobacter and Enterococcus strains added regularly to the rearing water from the postfertilized egg stage can become established in larval gastrointestinal tract. The Enterococcus strain was associated with increased larval growth, performance and microflora control, indicating its probiotic nature. SIGNIFICANCE AND IMPACT OF THE STUDY: Regular application of autochthonous probionts may promote larval welfare, development and stress tolerance at early stages, hence increasing production yield in intensive cod larviculture.

Isolation of putative probionts from cod rearing environment.

Survival problems are encountered at early stages of intensive fish rearing and antibiotics are widely used to remedy the situation. Probiotics may provide a potential alternative method to protect larvae from opportunistic and pathogenic bacteria and promote a balanced environment. This study was designed to search for new probiotics to target this critical period in cod rearing. Potential probionts were selected from the natural microbiota of cod aquacultural environment. The selection was based on several criteria: pathogen inhibition potential, growth characteristics, strain identification, metabolite production and adhesion to fish cell lines. Our study demonstrated that 14% of screened bacteria (n=188) had antagonistic properties towards fish pathogens. The majority of these isolates were Gram-positive (81%), belonging to Firmicutes (69.2%) and Actinobacteria (11.5%) phyla based on 16S rRNA gene sequencing. Only 6 (3.2%) of 188 isolates could inhibit all three pathogens tested: Vibrio anguillarum, Aeromonas salmonicida subsp. achromogenes and Vibrio salmonicida. Differences observed in activity intensity and spectrum among inhibitory isolates emphasise the need to develop probiotic mixtures for efficient prophylactic methods. Comparison of growth behaviour of inhibitory isolates and pathogens at cod rearing temperatures, metabolite production and adhesion capacity were considered for final probiont selection. Four promising isolates that could be used as a mixed supplement to rearing water were identified as putative probiotic bacteria. This study emphasises the importance and potential of lactic acid bacteria in aquaculture.

Influence of storage temperature on microbial spoilage characteristics of haddock fillets (Melanogrammus aeglefinus) evaluated by multivariate quality prediction.

The proliferation of specific spoilage organisms (SSO) and quality changes were evaluated in haddock fillets stored in styrofoam boxes at 0, 7 and 15 degrees C and under temperature fluctuations. A rapid electronic nose technique was used to monitor different classes of compounds, representing microbial metabolites that were characteristic for the onset of spoilage odors. Photobacterium phosphoreum predominated among the spoilage bacteria and high levels of TVB-N were observed at sensory rejection. Pseudomonas spp. appeared to be responsible for the development of sweet, fruity spoilage odors in haddock fillets coinciding with increasing response of the electronic nose CO sensor. H(2)S-producing bacteria, most likely Shewanella putrefaciens, were associated with the H(2)S sensor's response at abusive temperature conditions. Partial Least Squares Regression (PLSR) was used as an explorative tool to provide a better understanding of the spoilage potential of SSOs, by evaluating models based on electronic nose responses and counts of specific spoilage organisms to predict sensory quality (Torry scores). The best prediction of the sensory quality was obtained by PLSR models based on five variables: the electronic nose sensors (CO, NH(3) and H(2)S), pseudomonads counts and a time-temperature variable. Good agreement between the predicted and experimental data indicates that these variables characterize the sensory quality of haddock fillets stored under different temperatures.

Biopreservation of Brined Shrimp (Pandalus borealis) by Bacteriocins from Lactic Acid Bacteria.

In brined shrimp (ca. 3% NaCl), the effects of three different lactic acid bacteria bacteriocins (crude [6.54 x 10(sup10) U of bacteriocin activity {BU}/g] and purified [8.13 x 10(sup23) BU/g] nisin Z, carnocin UI49 [2.32 x 10(sup4) BU/g], and crude bavaricin A [2.78 BU/g]) on bacterial growth and shelf life were compared with those of a benzoate-sorbate solution (0.1% each [wt/wt]) and a control with no preservatives. The shelf life of shrimp subjected to the control treatment was found to be 10 days. Carnocin UI49 did not extend the shelf life, while crude bavaricin A (a cell-free supernatant of Lactobacillus bavaricus MI 401) resulted in a shelf life of 16 days, as opposed to 31 days with nisin Z for both its crude and purified forms. The benzoate-sorbate solution preserved the brined shrimp for the whole storage period (59 days). In the control, carnocin UI49, and crude bavaricin A treatments, a gram-positive flora dominated towards the end of the storage period while in the nisin Z treatment a gram-negative flora was more pronounced.