Preservation status and microbial communities of vacuum-packed hot smoked rainbow trout fillets.

Vacuum-packed hot smoked rainbow trout fillets from two different smokehouses of Greece were stored at 2 and 7.9 °C. Microbiological, sensory, and physicochemical changes were monitored. Microbial communities grown on MRS of three different pHs (5.4, 6.4 and 7.4) were also classified and identified using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Shelf-life was found to differ between products from the two smokehouses (A: 104 and 45 days, B: 100 and 45 days, at 2 and 7.9 °C, respectively). At the time point that sensory rejection was recorded, counts on MRS were found at higher population levels than the other microorganisms tested, almost in all cases. Out of the 567 colonies isolated from MRS of three different pHs, 71 classified as Enterococcus spp., 383 as Candida spp. and 113 as Lactobacillus spp.. Candida zeylanoides dominated exclusively in fillets from the smokehouse A during storage at 2 °C, while Lactobacillus sakei dominated clearly against C. zeylanoides at 7.9 °C, in all pH values. For the smokehouse B, C. zeylanoides or Enterococcus faecalis found to dominate initially in MRS of three pHs, C. zeylanoides, and/or Candida famata in the middle and/or the time point that sensory rejection was recorded at 2 °C, while Lactobacillus curvatus or E. faecalis at 7.9 °C. This study reveals the predominant cultivable spoilage microbiota of vacuum-packed hot smoked rainbow trout, and provides valuable information to the researcher and producers towards the production of more stable products with improved shelf-life.

HRM analysis as a tool to facilitate identification of bacteria from mussels during storage at 4 °C.

High-resolution melting (HRM) analysis followed by sequencing was applied for determination of bacteria grown on plates isolated from farmed mussels (Mytilus galloprovincialis) during their storage at 4 °C. The V3-V4 region of the 16S rRNA gene from the isolates was amplified using 16S universal primers. Melting curves (peaks) and high resolution melting curves (shape) of the amplicons and sequencing analysis were used for differentiation and identification of the isolated bacteria, respectively. The majority of the isolates (a sum of 101 colonies, from five time intervals: day 0, 2, 4, 6 and 8) from non-selective solid medium plates were classified in four bacterial groups based on the melting curves (peaks) and HRM curves (shape) of the amplicons, while three isolates presented distinct HRM curve profiles (single). Afterwards, sequencing analysis showed that the isolates with a) the same melting peak temperature and b) HRM curves that were >95% similar grouped into the same bacterial species. Therefore, based on this methodology, the cultivable microbial population of chill-stored mussels was initially dominated by Psychrobacter alimentarius against others, such as Psychrobacter pulmonis, Psychrobacter celer and Klebsiella pneumoniae. P. alimentarius was also the dominant microorganism at the time of the sensory rejection (day 8). Concluding, HRM analysis could be used as a useful tool for the rapid differentiation of the bacteria isolated from mussels during storage, at species level, and then identification is feasible by the sequencing of one only representative of each bacterial species, thus reducing the cost of required sequencing.

Bacterial communities and potential spoilage markers of whole blue crab (Callinectes sapidus) stored under commercial simulated conditions.

Bacterial communities composition using 16S Next Generation Sequencing (NGS) and Volatile Organic Compounds (VOCs) profile of whole blue crabs (Callinectes sapidus) stored at 4 and 10 °C (proper and abuse temperature) simulating real storage conditions were performed. Conventional microbiological and chemical analyses (Total Volatile Base-Nitrogen/TVB-N and Trimethylamine-Nitrogen/TMA-N) were also carried out. The rejection time point was 10 and 6 days for the whole crabs stored at 4 and 10 °C, respectively, as determined by development of unpleasant odors, which coincided with crabs death. Initially, the Aerobic Plate Count (APC) was 4.87 log cfu/g and increased by 3 logs at the rejection time. The 16S NGS analysis of DNA extracted directly from the crab tissue (culture-independent method), showed that the initial microbiota of the blue crab mainly consisted of Candidatus Bacilloplasma, while potential pathogens e.g. Listeria monocytogenes, Pseudomonas aeruginosa and Acinetobacter baumannii, were also found. At the rejection point, bacteria of Rhodobacteraceae family (52%) and Vibrio spp. (40.2%) dominated at 4 and 10 °C, respectively. TVB-N and TMA-N also increased, reaching higher values at higher storage temperature. The relative concentrations of some VOCs such as 1-octen-3-ol, trans-2-octenal, trans,trans-2,4-heptadienal, 2-butanone, 3-butanone, 2-heptanone, ethyl isobutyrate, ethyl acetate, ethyl-2-methylbutyrate, ethyl isovalerate, hexanoic acid ethyl ester and indole, exhibited an increasing trend during crab storage, making them promising spoilage markers. The composition of microbial communities at different storage temperatures was examined by 16S amplicon meta-barcoding analysis. This kind of analysis in conjugation with the volatile profile can be used to explore the microbiological quality and further assist towards the application of the appropriate strategies to extend crab shelf-life and protect consumer's health.

Microbial spoilage investigation of thawed common cuttlefish (Sepia officinalis) stored at 2 °C using next generation sequencing and volatilome analysis.

Cephalopods are highly appreciated with increasing demand seafood, but are also very perishable and deteriorate fast mainly due to microbiological spoilage. For this reason exploration of bacterial communities through 16S Next Generation Sequencing (NGS) and Volatile Organic Compounds (VOCs) analysis was performed. Furthermore, sensory evaluation, classical microbiological analysis, Total Volatile Base-Nitrogen/TVB-N and Trimethylamine-Nitrogen/TMA-N determination were also carried out. Shelf-life of thawed cuttlefish (Sepia officinalis) stored at 2°C determined by sensory evaluation was 4 days. Aerobic Plate Counts (APC) reached the levels of 6.6 log cfu/g. The initial and final population of all spoilage microorganisms enumerated with selective media was under detectable levels with the exception of Pseudomonas. Based on 16S NGS analysis, Psychrobacter were the dominants among others, e.g. Pseudomonas, Shewanella, Comamonas, Carnobacterium, Vagococcus, of the initial microbiota. Psychrobacter was also the dominant microorganisms of the spoiled cuttlefish. TVB-N and TMA-N increased considerably only at the late stages of storage. A plethora of VOCs were produced and some exhibited an increasing profile throughout storage, making them promising molecules as freshness indicators in contrast to TVB-N and TMA-N. The application of next generation sequencing revealed the microbiota that escapes the classic microbiological methodologies, showing that other microorganisms different from those determined on selective culture media might be the main cause of microbiological spoilage.

Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis.

Investigation of the initial and spoilage microbial diversity of iced stored sea bream was carried out. Culture dependent methods were used for bacterial enumeration and phenotypic identification of bacterial isolates, while culture independent methods, using bacterial 16S rRNA gene amplification, cloning and sequencing of DNA extracted directly from the flesh were also employed. The culture dependent approach revealed that the initial microbiota was dominated by Acinetobacter, Shewanella, Pseudomonas and Flavobacterium, while at the end of shelf-life determined by sensory analysis (16 days), the predominant microbiota was Pseudomonas and Shewanella. Culture independent approach showed that initially the sea bream flesh was strongly dominated by Acinetobacter, while Pseudomonas, Aeromonas salmonicida and Shewanella were the predominant phylotypes at the end of shelf-life. Initial and spoilage microbiota comprised of phylotypes previously identified by others using traditional or molecular techniques. However, Aeromonas has not been reported as part of the dominant microbiota of sea bream at the time of spoilage. Combination of classical and molecular methodologies better reveals the microbiota during storage by revealing bacteria that escape standard approaches and, thus, provides valuable complementary information regarding microbiological spoilage.

Effect of Satureja thymbra essential oil on growth-no growth interfaces of Listeria monocytogenes Scott A and Salmonella Enteritidis PT4, at various temperatures, pH, and water activities.

Antimicrobial efficacy of Satureja thymbra essential oil against Listeria monocytogenes Scott A and Salmonella ser. Enteritidis PT4 was evaluated in vitro by modeling the growth boundaries at various temperatures, pH, and NaCl concentrations. Growth-no growth (turbidity) data were modeled by logistic polynomial regression. The concordance indices and the Hosmer- Lemeshow statistics of both logistic models indicated a good fit to the observed data. Salmonella Enteritidis was more sensitive at increasing salt content as compared with L. monocytogenes. On the other hand, pH changes had greater effect on growth initiation of L. monocytogenes than they had on growth initiation of Salmonella Enteritidis. Presence of essential oil up to 0.06% (vol/vol) had no or little effect on growth initiation of both microorganisms tested, while the concentration of 0.1% (vol/vol) essential oil exhibited great inhibition on growth initiation, especially when it was combined with increased salt content and low temperatures. The antimicrobial potency of S. thymbra essential oil was more pronounced when multiple hurdles were applied. Modeling the growth boundaries offers a useful tool to food microbiologists for assessing the antimicrobial activity in a range of food preservation conditions as compared with the conventional MIC determination.

Effect of NaCl and KCl on fate and growth/no growth interfaces of Listeria monocytogenes Scott A at different pH and nisin concentrations.

AIMS: The fate of Listeria monocytogenes Scott A, was studied in broth, at different a(w)s (by adding NaCl or KCl from 0.0 to 1.4 mol l(-1)), pHs (from 4.0 to 7.3 by adding lactic acid), and nisin concentrations (from 0 to 100 IU ml(-1)). METHODS AND RESULTS: Increasing salt and nisin concentrations and decreasing pH resulted in lower growth rates and extended lag phases. At pH 4.5 no growth was observed while in presence of nisin and/or 1 mol l(-1) salts of both kinds, L. monocytogenes Scott A was inactivated. Equal-molar concentrations of NaCl or KCl (similar a(w)), exerted similar effects against L. monocytogenes in terms of lag phase duration, growth or death rate. The growth boundaries of L. monocytogenes Scott A at 5 degrees C were also estimated by growth/no growth turbidity data, modeled by logistic polynomial regression. The concordance of logistic models, were 99.6 and 99.8% for NaCl and KCl, respectively. CONCLUSIONS: The growth interfaces derived by both NaCl and KCl models were almost identical. Hence, NaCl can be replaced by KCl without risking the microbiological safety of the product. Increasing nisin concentrations markedly affected the interface resulting in a more inhibitory environment for L. monocytogenes Scott A. Low to medium salt concentrations (0.3-0.7 mol l(-1) of either NaCl or KCl) provided a protective effect against inhibition of L. monocytogenes Scott A by nisin. SIGNIFICANCE AND IMPACT OF THE STUDY: Modelling the growth boundaries not only contributes to the development of safer food by providing useful data, but can also be used to study interactions between factors affecting initiation of growth of pathogenic micro-organisms.

Effect of nisin on growth boundaries of Listeria monocytogenes Scott A, at various temperatures, pH and water activities.

The effect of nisin on growth boundaries of Listeria monocytogenes Scott A in Tryptone Soy Broth (TSB) under different a(w)s, pH, and temperatures was studied. Growth/no growth turbidity data was modeled using logistic regression. Combinations of various temperatures (5-35 degrees C), pH (4.05-6.70) adjusted with HCl, a(w)s (0.937-0.998) NaCl (0.5-10.5%) and nisin (0-100 IU/ml) were used to monitor the growth/no growth response of L. monocytogenes Scott A for 60 days. The concordance of the logistic regression model was 99.4%, indicating successful data fitting. The minimum pH at which growth was observed was 4.81 at the temperature range of 25-35 degrees C and at a(w) as high as 0.992. Growth was observed at a(w) as low as 0.937, at pH 6.7, at the temperature range of 25-35 degrees C. Increasing nisin concentrations above 25 IU/ml resulted in a more inhibitory environment for L. monocytogenes. Presence of 100 IU/ml resulted in a minimum pH for growth at 5.20, and a minimum a(w) at 0.967 at the temperature range of 25-35 degrees C. It was remarkable that low to medium salt concentrations (2.5-4.5 NaCl% w/v) provided a protective effect against inhibition of L. monocytogenes by nisin. The present study points out the applicability of growth/no growth modeling in order to study any interactions between various factors affecting initiation of growth of micro-organisms, in which its turn helps the understudying of microbe-food ecosystem relations and the development of safer food.

Temperature shock, injury and transient sensitivity to nisin in Gram negatives.

AIMS: The effect of thermal stresses on survival, injury and nisin sensitivity was investigated in Salmonella Enteritidis PT4, PT7 and Pseudomonas aeruginosa. METHODS AND RESULTS: Heating at 55 degrees C, rapid chilling to 0.5 degrees C or freezing at -20 degrees C produced transient sensitivity to nisin. Cells were only sensitive if nisin was present during stress. Resistance recovered rapidly afterwards, though some cells displayed residual injury. Injury was assessed by SDS sensitivity, hydrophobicity changes, lipopolysaccharide release and NPN uptake. LPS release and hydrophobicity were not always associated with transient nisin sensitivity. Uptake of NPN correlated better but persisted longer after treatment. CONCLUSIONS: Thermal shocks produce transient injury to the outer membrane, allowing nisin access. After treatment, the permeability barrier is rapidly restored by a process apparently involving reorganization rather than biosynthetic repair. SIGNIFICANCE AND IMPACT OF THE STUDY: Inclusion of nisin during food treatments that impose sub-lethal stress on Gram negatives could increase process lethality, enhancing microbiological safety and stability.

Transient sensitivity to nisin in cold-shocked Gram negatives.

Rapid chilling in the presence of nisin caused a dose-dependent reduction in the populations of several Gram-negative bacteria, despite the fact that appreciable structural injury to the outer membrane was not detected. Pseudomonas aeruginosa was most affected, followed by Pseudomonas fragi, Salmonella enteritidis PT4, PT7 and Escherichia coli, respectively. Addition of nisin after the chilling treatment had no effect. The results are ascribed to a transient susceptibility caused by phase changes in the lipids associated with the outer membrane, which are rapidly reversed when the cells return to higher temperatures. Combinations of chilling shock, nisin and EDTA gave much lower reductions of Salmonella and Pseudomonas on chicken skin in comparison with broths. This is attributed to a buffering of the temperature shock experienced by adherent bacteria and binding of the nisin by food particles.

Effect of chelators and nisin produced in situ on inhibition and inactivation of gram negatives.

The ability of chelators and nisin generated in situ to inhibit and inactivate E. coli and other gram negatives in a model substrate was investigated. The effect of various chelators and different concentrations of exogenous nisin on inhibition of E. coli in broth medium showed that only EDTA and pyrophosphates were able to cause appreciable inhibition of E. coli by nisin. In a broth where L. lactis NCFB 497 produced nisin in a concentration of 250-300 IU/ml, pyrophosphates were unable to inactivate E. coli. Under the same conditions, addition of EDTA led to inactivation of E. coli at neutral and slightly acidic pH only. A cocktail of strains of E. coli was less sensitive than E. coli ATCC 25922 alone. Pseudomonas aeruginosa was more sensitive and salmonellae more resistant. EDTA also caused a slight reduction in the L. lactis population and its biochemical activity as regards pH drop and acid production. Some of the inhibition of E. coli could be ascribed to the physical presence of Lactococcus cells rather than their metabolites excreted into the medium. Failure to observe any inhibition in fermented broths at their natural pH (4.0) was ascribed to the poor chelating power of EDTA under acid conditions.

Effect of nisin on heat injury and inactivation of Salmonella enteritidis PT4.

The ability of heat injury to confer sensitivity to nisin in a Gram negative pathogen was investigated. Injury and inactivation kinetics of Salmonella enteritidis PT4 in the presence of nisin were determined in media, liquid whole egg and egg white using cultural methods and capacitance monitoring to detect injury. Addition of nisin in concentrations from 500 IU/ml to 2500 IU/ml in the heating menstruum caused a reduction of required pasteurisation time of up to 35%, principally as a result of its effect on cells suffering damage during heating. In egg white and liquid whole egg the organism's heat susceptibility was greater than in nutrient broth, particularly in egg white which contained no fat and had an alkaline pH. The effect of nisin on heat susceptibility was however less pronounced than in nutrient broth due to its interaction with protein and fat. Though nisin did not enhance the lethality of heat processes, injury is more severe in egg white containing nisin, presumably as a result of its interaction with antimicrobial factors in egg white.