Ioannis S. Boziaris

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 aws (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).

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.

Microbiological spoilage and investigation of volatile profile during storage of sea bream fillets under various conditions.

Volatile organic compound (VOC) profile was determined during storage of sea bream (Sparus aurata) fillets under air and Modified Atmosphere Packaging (MAP - CO2/O2/N2: 60/10/30) at 0, 5 and 15°C. Microbiological, TVB-N (Total Volatile Base Nitrogen) and sensory changes were also monitored. Shelf-life of sea bream fillets stored under air was 14, 5 and 2days (d) at 0, 5 and 15°C respectively, while under MAP was 18, 8, and 2d at 0, 5 and 15°C respectively. At the end of shelf life, the total microbial population ranged from 7.5 to 8.5logcfu/g. Pseudomonas spp. were among the dominant spoilage microorganisms in all cases, however growth of Brochothrix thermosphacta and Lactic Acid Bacteria (LAB) were favoured under MAP compared to air. TVB-N production was favoured at higher temperatures and under air compared to lower temperatures and MAP. TVB-N increased substantially from the middle of storage and its value never reached concentrations higher than 30-35mgN/100g, which is the legislation limit, making it a poor chemical spoilage index (CSI). A lot of alcohols, aldehydes, ketones and ethyl esters that were detected in the present study have been reported as bacterial metabolites, others as products of chemical oxidation while others as aroma constituents. VOCs such as 3-methylbutanal, acetic acid, ethanol, ethyl esters of isovaleric and 2-methylbutyric acids, 1-penten-3-ol, 1-octen-3-ol and cis-4-heptenal appeared from the early or middle stages and increased until the end of storage. From those only 3-methylbutanal, acetic acid, ethanol and the ethyl esters have been reported as microbial origin, making them potential CSI candidates of sea bream fillets.

Bacterial Synergism or Antagonism in a Gel Cassette System

ABSTRACT The growth and the metabolic activity of Shewanella putrfaciens, Brochothrix thermosphacta, and Pseudomonas sp., when cultured individually or in all possible combinations in gel cassettes system supplemented with 0.1% glucose at 5°C, were investigated. The overall outcome was that the coexistence of the above-mentioned microorganisms affected not only each growth rate but also their type of metabolic end products compared to the control cultures. These effects were varied and depended on the selection of the combination of the tested bacteria. For example, the growth of Pseudomonas sp. strains cocultured with either B. thermosphacta or S. putrefaciens strains resulted in different effects: a promoting one for the first and an inhibitory one for the second. Moreover, the production of formic acid and two unidentified organic acids (peaks a and b) was characteristic in all cases in which S. putrefaciens was cultured.

Microbiological changes, shelf life and identification of initial and spoilage microbiota of sea bream fillets stored under various conditions using 16S rRNA gene analysis.

BACKGROUND Sea bream fillets are one of the most important value-added products of the seafood market. Fresh seafood spoils mainly owing to bacterial action. In this study an exploration of initial and spoilage microbiota of sea bream fillets stored under air and commercial modified atmosphere packaging (MAP) at 0 and 5 °C was conducted by 16S rRNA gene sequence analysis of isolates grown on plates. Sensory evaluation and enumeration of total viable counts and spoilage microorganisms were also conducted to determine shelf life and bacterial growth respectively. RESULTS Different temperatures and atmospheres affected growth and synthesis of spoilage microbiota as well as shelf life. Shelf life under air at 0 and 5 °C was 14 and 5 days respectively, while under MAP it was 20 and 8 days respectively. Initial microbiota were dominated by Pseudomonas fluorescens, Psychrobacter and Macrococcus caseolyticus. Different temperatures and atmospheres affected the synthesis of spoilage microbiota. At the end of shelf life, different phylotypes of Pseudomonas closely related to Pseudomonas fragi were found to dominate in most cases, while Pseudomonas veronii dominated in fillets under MAP at 0 °C. Furthermore, in fillets under MAP at 5 °C, new dominant species such as Carnobacterium maltaromaticum, Carnobacterium divergens and Vagococcus fluvialis were revealed. CONCLUSION Different temperature and atmospheric conditions affected bacterial growth, shelf life and the synthesis of spoilage microbiota. Molecular identification revealed species and strains of microorganisms that have not been reported before for sea bream fillets stored under various conditions, thus providing valuable information regarding microbiological spoilage.

Microbiological aspects and shelf life of processed seafood products

BACKGROUND Fresh fish and seafoods are very perishable products mainly owing to microbial activity of specific spoilage micro-organisms. Application of hurdle technology leads to a variety of processed products with extended shelf life. In this study, sensory evaluation and microbiological analysis were carried out on 17 processed seafood products stored at 4 °C to determine their shelf life and the predominant spoilage micro-organisms. RESULTS Shelf life determined by sensory analysis varied from 66 to 180 days depending on the product. The cause of spoilage for most of the products was the development of off-flavours/off-odours, while two products were rejected owing to oil discolouration. Pseudomonads were in most cases below detection limit. H₂S-producing bacteria, Brochothrix thermosphacta and Enterobacteriaceae were below detection limit throughout the experiment. The predominant spoilage micro-organisms were lactic acid bacteria and yeasts. Hygiene indicators such as Staphylococcus spp. and total coliforms were also below detection limit in all samples. CONCLUSION Primarily the initial pH and secondarily the NaCl content determined shelf life duration. Under the applied conditions, lactic acid bacteria and yeasts predominated. The contribution of chemical oxidation and/or autolysis to spoilage and shelf life might be important for most of the products.

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.

Dynamics of biofilm formation by Listeria monocytogenes on stainless steel under mono-species and mixed-culture simulated fish processing conditions and chemical disinfection challenges

The progressive ability of a six-strains L. monocytogenes cocktail to form biofilm on stainless steel (SS), under fish-processing simulated conditions, was investigated, together with the biocide tolerance of the developed sessile communities. To do this, the pathogenic bacteria were left to form biofilms on SS coupons incubated at 15°C, for up to 240h, in periodically renewable model fish juice substrate, prepared by aquatic extraction of sea bream flesh, under both mono-species and mixed-culture conditions. In the latter case, L. monocytogenes cells were left to produce biofilms together with either a five-strains cocktail of four Pseudomonas species (fragi, savastanoi, putida and fluorescens), or whole fish indigenous microflora. The biofilm populations of L. monocytogenes, Pseudomonas spp., Enterobacteriaceae, H2S producing and aerobic plate count (APC) bacteria, both before and after disinfection, were enumerated by selective agar plating, following their removal from surfaces through bead vortexing. Scanning electron microscopy was also applied to monitor biofilm formation dynamics and anti-biofilm biocidal actions. Results revealed the clear dominance of Pseudomonas spp. bacteria in all the mixed-culture sessile communities throughout the whole incubation period, with the in parallel sole presence of L. monocytogenes cells to further increase (ca. 10-fold) their sessile growth. With respect to L. monocytogenes and under mono-species conditions, its maximum biofilm population (ca. 6logCFU/cm2) was reached at 192h of incubation, whereas when solely Pseudomonas spp. cells were also present, its biofilm formation was either slightly hindered or favored, depending on the incubation day. However, when all the fish indigenous microflora was present, biofilm formation by the pathogen was greatly hampered and never exceeded 3logCFU/cm2, while under the same conditions, APC biofilm counts had already surpassed 7logCFU/cm2 by the end of the first 96h of incubation. All here tested disinfection treatments, composed of two common food industry biocides gradually applied for 15 to 30min, were insufficient against L. monocytogenes mono-species biofilm communities, with the resistance of the latter to significantly increase from the 3rd to 7th day of incubation. However, all these treatments resulted in no detectable L. monocytogenes cells upon their application against the mixed-culture sessile communities also containing the fish indigenous microflora, something probably associated with the low attached population level of these pathogenic cells before disinfection (<102CFU/cm2) under such mixed-culture conditions. Taken together, all these results expand our knowledge on both the population dynamics and resistance of L. monocytogenes biofilm cells under conditions resembling those encountered within the seafood industry and should be considered upon designing and applying effective anti-biofilm strategies.

Microbiological quality of raw and processed wild and cultured edible snails

BACKGROUND An increasing interest in snail farming in Greece and other European countries has been observed. Despite the fact that edible snails have been involved with problems of Salmonella spp. contamination, there are to our knowledge only limited studies regarding microbiological safety and hygiene of such products. Enumeration of microbial populations and presence/absence of Salmonella spp. in snail meat and intestines of wild Cornu aspersum, Helix lucorum and cultured Cornu aspersum snails from indoor/outdoor type farms was conducted. Furthermore, snail-processing steps were simulated in the laboratory and the population reduction in snail meat was determined. RESULTS Microbial populations were higher in intestines than snail meat in almost all cases. Escherichia coli/coliforms and Enterococcus spp. populations were lower in the intestines and snail meat of cultured C. aspersum. Salmonella spp. were detected in the intestines and snail meat of wild snails only. The high levels of bacterial populations were considerably reduced after the appropriate processing. CONCLUSION The lower populations of E. coli/coliforms, Enterococcus spp. and especially the absence of Salmonella spp. in cultured snails show that the controlled conditions decrease the possibility of pathogen presence and contribute to food safety and public health.

A meta-barcoding approach to assess and compare the storage temperature-dependent bacterial diversity of gilt-head sea bream (Sparus aurata) originating from fish farms from two geographically distinct areas of Greece

Bacterial diversity of whole gilt-head sea bream (Sparus aurata L. 1758) originating from Ionian and Aegean Sea aquaculture farms and stored at 0 (ice), 4 and 8 °C was determined by 16S rRNA gene amplicon sequencing method using the Illuminas MiSeq platform. The composition of Aerobic Plate Counts (APC) was also monitored by 16S rRNA gene sequencing. The rejection time point of sea bream from either area, as determined by sensory evaluation, was about 14, 6 and 3 days at 0, 4 and 8 °C, respectively. APC was approximately 4.5 log cfu/g at day 0 and ranged from 7.5 to 8.5 log cfu/g at sensory rejection. Culture-depended analysis showed that Pseudomonas and Shewanella were the most abundant microorganisms grown on plates for both seas. Moreover, culture-independent analysis of DNA extracted directly from fish flesh showed that sea bream originating from different geographical areas exhibited different bacterial diversity. Pseudomonas and Psychrobacter were the dominant microorganisms of chill-stored fish from Ionian (apart from 8 °C, where Carnobacterium dominated) and Aegean Sea, respectively. In addition, small changes of storage temperature greatly affected bacterial microbiota of stored fish. Various bacterial species, not detected by conventional microbiological methods, were also revealed through 16S amplicon sequencing. In conclusion, the use of NGS approach is a promising methodology for assessing bacterial diversity of sea bream originating from different geographical areas and stored at various temperatures.